Studies of Actomyosin from Cardiac Muscle of Dogs withExperimental Congestive Heart FailureBy JAMES 0. DAVIS, P H . D . , M.D., MARY TBAPASSO, M.S., AND

NICHOLAS A. YANKOPOULOS, M.D.

With the surgical assistance of Alfred Casper

Comparative studies wore made on cardiac actomyosin from normal dog's and .from dogswith experimental heart failure. Actomyosin was characterized by ultracentrifugal sedi-mentation velocity, viscosity and ATP-ase measurements. The data on actomyosin fromnormal cardiac muscle showed a striking' similarity to the findings reported by othersfor skeletal muscle actomyosin. The only difference found between cardiac actomyosinfrom the normal and experimental material was an abnormal component (SJOW = 5.0-6.7)iu the sedimentation pattern for actomyosin from 4 of 11 dog's with cardiac failure. Itseems likely that the changes in actomyosin which resulted in the abnormal sedimentationpattern were produced during- extraction or preparation of the actomyosin and that theydo not reflect an altered state of actomyosin in the functioning heart. The explanationfor the occurrence of this slow sedimentation component solely in the experimentalmaterial is not clear.

THE nature of the biochemical changesin the failing heart is one of the im-

portant unsolved problems in cardiovascularphysiology and disease. Normal myocardialfunction is dependent upon a series of bio-chemical and biophj'sical changes whichterminate in cardiac contraction. Myocardialfailure may result as a consequence of inter-ruption of these sequential changes at somecritical stage. At an orgauismal level, a de-crease in stroke work per unit of end dias-folie volume occurs and the well-known syn-drome of congestive heart failure ensues.

Many aspects of the biochemical changesleading to contraction of cardiac muscle havebeen investigated. Biug,1 Goodale2 and Olson3

have studied the uptake of substrate and ofoxygen by the failing heart but they foundno evidence of a defect. Also, studies of thehigh energy phosphate compounds (adeno-sine triphosphate and phosphocreatine) inthe myocardium of failing heart-lung prep-parations by Wollenberger4 aud in hearts ofdogs with chronic low output failure by

From the Section on Experimental CardiovascularDisease, Laboratory of Kidney and Electrolyte Me-tabolism, National Heart Institute, Bcthesda, Md.

"Received for publication Juno 16, 1959.

Olson and associates8 have failed to demon-strate a depletion of these sources of energy.Instead, available evidence has favored theview that a biochemical defect is presentin the assimilation of phosphate bond energyby the contractile proteins, or in actomyosinitself.

Attention has been directed, therefore, atthe contractile proteins in the failing myo-cardium. Benson and co-workers5"7 reportedthat actomyosin is partially broken down intoactin and uncombined myosin. Olson and as-sociates8 have suggested that myosin fromthe failing heart has a molecular weight of500,000 to 750,000 which is approximately2 to 3 times the value they found for normalcardiac myosin. Both groups of workers madeno distinction between contractile proteins ob-tained from the right and the left ventriclesof dogs with right-sided lesions of tricus-pid insufficiency and pulmouic stenosis.Kako and Bing9 reported that the contrac-tility of actomyosin bands from failing heartmuscle of humans was reduced, but theyfailed to mention whether the actomyosincame from the right or the left ventricle, andsome of their patients had hypertensive heartdisease.

957 Circulation Research, Volume VII, November i959

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958 DAVIS, TRAPASSO, YANKOPOULOS

The present observations were undertakento characterize cardiac actomyosin from nor-mal dogs and from dogs with experimentalheart failure by ultracentrifugal sedimenta-tion velocity, viscosity and adenosine triphos-phatase (ATP-ase) measurements. Physio-logic studies were made throughout the clini-cal course of heart failure to define as com-pletely as possible the physiologic state as-sociated with the pathogenesis of cardiacfailure and to determine changes secondaryto failure which might conceivably alter thecardiac muscle proteins. The principal partof the physiologic data is presented else-where.10

METHODS

Cardiac muscle was obtained from 15 normaldogs, 7 dogs with cardiac failure produced by con-trolled progressive pulmonic stenosis,11 5 dogswith chronic congestive failure secondary to trieus-pid insufficiency and pulmonic stenosis,1- and 3dogs with chronic ascites produced by thoracic in-ferior vena cava constriction.13 On the day ofsacrifice, physiologic studies were made to define(1) the degree of sodium retention, (2) the quan-tity of ascites present, (3) mean right atrial pres-sure (RAP), and (4) left ventricular end diastolicpressure. The methods are described elsewhere.10

To obtain the cardiac muscle, the animals wereanesthetized lightly with intravenous sodium pento-barbital. The chest was opened quickly, the heartexcised and washed with cold water, and placedin a container with chipped ice. All samples weretaken from the right and left ventricular walls.The muscle was cut into small pieces of approxi-mately 0.1 Gin. from which 1.0 Gm. (0.95 to 1.05)samples were weighed to within 0.1 mg. Immedi-ately after weighing, the muscle samples wereplaced in a cold room (0 to 5 C) . All muscle re-ferred to hereafter as fresh was homogenized andextraction begun immediately; the remaining sam-ples were frozen at —20 C.

Homogenization and extraction of cardiac musclefor actomyosin were performed at 0 to 5 C. Theextraction procedure is the same as that employedby Benson.5"7 One gram samples were homoge-nized in 7 ml. of Weber's solution (0.6 M KC1.0.04 M NaHC03 and 0.01 M Na2CO3; pH = 9.3at 10 C.) in Potter tissue grinders (40 ml.) byhand until all visible strands of muscle and con-nective tissue disappeared. This procedure wasadopted after failure to obtain complete homogeni-zation with several different types of motor driven

grinders. Seven milliliters of 0.6 M KC1 and 2mg. of adenosine triphosphate (ATP) were addedto the homogenate and extraction carried out for20 to 24 hours.

Following extraction, 15 ml. of 0.6 M KC1 wen;added to the homogenate which was then centii-fuged at 2000 g. for 10 inin. in a Lourdes modelAT centrifuge. After decanting, the supernatantwas centrifuged for an additional 15 min. at2000 g. The resultant preparation will be calledthe soluble extract. For preparation of actomyo-sin, 5 volumes of glass distilled water were addedto the soluble extract. The solution was adjustedto pH 6.8 with 0.5 X acetic acid; actomyosin ap-peared as a flocculent precipitate. Actomyosin wasseparated by centrifugation (S000 g. for 10min.), washed with glass distilled water, and dis-solved in 0.6 M KC1.

To determine the yield of actomyosin, 7 ml.samples of soluble extract were subjected to theprocedure described. Two 1 Gin. samples from eachventricle were analyzed on fresh tissue; repeatanalyses were made on frozen tissue. Total nitro-gen was determined on the precipitated actomyo-sin by miero-Kjeldahl analysis and the resultmultiplied by 6 to obtain the amount of actomyo-sin. Nbnprotein nitrogen was determined on thetrichloroacetic acid filtrate of the soluble extract.The total protein content of heart muscle was ob-tained by determining the total nitrogen in 1 Gin.samples, subtracting the nonprotein nitrogen andmultiplying the result by 6.

The sedimentation velocity of cardiac actomyo-sin was studied with a model E Spinco analyticultracentrifuge. One preparation of actomyosinwas made from fresh cardiac muscle from eachdog and all subsequent preparations from eachanimal were obtained from frozen tissue. Cen-trifugation was carried out on the same day theprotein preparation was complete. Actomyosin wasdissolved in 0.6 M KC1 and a preliminary centrif-ugation at 20.000 g. for 20 min. was made in aLourdes model AT centrifuge. The sedimentationvelocity was determined on 2 samples simultan-eously by vise of a wedge cell and a plain cell.Kel F centerpieces were used to prevent denatura-tion of the protein. The routine procedure wasto centrifuge an actomyosin preparation in thewedge cell and the same actomyosin preparation,ATP and MgCL. in the plain cell. The final con-centrations of ATP and MsrClo were 5 X 10"3 Mand 10~3 M, respectively. An equivalent volume of0.6 M KC1 was added to the actomyosin preparationin the wedge cell so that the protein concentrationin the 2 cells was identical. By addition of ATP.actomyosin was converted to myosin and the sedi-mentation velocity of both proteins was measured

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CONTRACTILE PROTEINS IN HEART FAILURE 959

simultaneously. Ultraeentrifugation was performedat 59,780 RPM or 250,000 g. at an average temper-ature of 11.4 C. Values of 0.74 and 0.72S were usedfor the partial specific volumes of actomyosin andmyosin, respectively. In addition to these observa-tions, the effects of dialysis of aetoinyosin against0.1 M Na2CO3 for 24 hours at 0 to 5 C. werestudied by ultracentrifugation.

Viscosity was measured with size I and IIUbbelohde viscosiineters with outflow times for0.6 M KC1 of 16.8 and 100.0 sec, respectively.Two viscosiineters with different flow times wereused to measure viscosity at 2 widely differentmean velocity gradients" (75 to ]50 for size IIand .1,200 to 1,500 for size I) because moleculessuch as actomyosin with a high axial ratio shownon-Newtonian viscosity changes due to shearrate dependence. The use of 2 viseosimeters withdifferent moan velocity gradients does not pro-vide a completely satisfactory solution to theproblem of shear rate dependence but only com-parative data and not absolute values are ofprimary importance in this study.

All measurements of viscosity were made onactomyosin immediately after preparation fromfresh right ventricular muscle or skeletal muscle(the latter measurements were made to obtaincomparative data). The protein was dissolved in0.6 M KC1. Viscosity measurements with thesize I viscosimeter were made at 0 to 0.5 C. byplacing the viscosimeter in an ice bath and con-stantly stirring the ice. Studies with the size ITviscosimeter were made in a constant tempera-ture water bath at 22.5 ± .02 C. A phosphatebuffer system adjusted to pH of 7.6 was used.The relative viscosity (rj rel) was determined asthe ratio of outflow time for the solution of acto-myosin to that of 0.6 M KC1. "ATP sensitivity"was determined on actomyosin solutions of ap-proximately 1 mg./ml. This test is a means ofcharacterizing the actin content of actonryosin.11

It is made by measuring the relative viscosity ofthe actomyosin solution before and after additionof ATP. One tenth milliliter of 0.200 M ATPwas added to 11.1 ml. of protein solution. The"ATP sensitivity" is expressed in per cent by the

Zn—Z,,ATPfoi-inul. — i . X 100 where Zn is the

viscosity number and ZnATP is the viscositynumber after addition of ATP. The viscosity

*The mean velocity gradient P is defined as P =S V

whore V is the volume in millilitors of liquid3 K3tpassing through tho capillary, R is radius in cen-timeters of the capillary, and t is the flow time inseconds.

number Zn is obtained from the expression2.303 log v rel

Xn - d

where C is the concentration in grams per L. Thespecific viscosity, ?;S1I, is equal to 7;re|—1.

ATP-ase activity of actomyosin from the rightventricle was measured under conditions designedto yield maximal or near maximal activity.15

The reaction mixture contained 0.2 to 0.3 mg./ml.of actomyosin in 0.05 M KC1, either 0.1 M or0.01 M CaClo, 0.002 M ATP and 0.02 M Trisbuffer. The pH of the reaction mixture was ap-proximately 7.S. All reactions were carried outin a constant temperature bath at 22.5 ± .02 C.After incubation for 0, 2, 4, 6, S, or 10 miu., thereaction was stopped by addition of 1 ml. of 20per cent triehloroacetic acid. The nitrate wasanalyzed by the Fiske-SubbaRow method for in-organic phosphorus.

RESULTS

Physiologic State of Dogs at the Time ofSacrificeAll 7 animals with progressive pulmonic

stenosis showed evidence of right heart failureincluding a marked elevation in mean RAP,hepatomegaly, a very low rate of Na ex-cretion and ascites (table 1). Left ventricularend-diastolie pressure was measured in 2 ofthe dogs and found to be normal. Elsewhere10

measurements of filling pressure in the leftventricle showed normal values for 7 otherdogs with heart failure secondary to pro-gressive pulmonic stenosis. Additional dataon cardiovascular and renal hemodynamicfunction in this type of experimental heartfailure have been reported previously.11

The findings from studies of the dogs withchronic congestive failure secondary to tri-cuspid insufficiency and pulmonic stenosisare presented in table 1. There was evidenceof pure right-sided heart failure. Other dataare presented elsewhere.10

Actomyosin Content of Ventricular HeartMuscle. To exclude the influence of increasedwater content of the tissue from animalswith heart failure, the data are expressedas the per cent of actomyosin per grain oftotal protein. The yields of actomyosiu were(1) 28.7 ± 3.1* for normal right ventricle

"Standard deviation.

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960 DAVIS, TRAPARSO, YANKOPOULOS

TABLE 1.—Physiologic Data at Sacrifice

Dog

Cardiac1o

3

45

6

7

Cardiac

1O

3

4

5

Time afterlast operation

(Days)

failure secondary

14

27

48

IS19

3025

failure produced26

.119

91

U S

195

Thoracic inferior vena ct1

3

92

74166

Duration of Volume ofascites ascites(Days) (L.)

E

• to pulnionic

44

5

3

3

9

12

by tricuspid24

114

SO

SS

192

stenosis0.3

1.8

+t1.2

-f++

insufficiency3.0

0.1

S.O

0.24.9

iva constrictionSS

61

165

3.0

1.5

2.5

Renal Naexcretion*

(mEa./day)E

4.3

4.4

1.2

1.6

1.2

6.4

2.2

and pulnionic

5.S17.2

4.3

15.4

39.0

1.9

7.50.6

Mean rightatrial pressure!

(mm. water)C E

—

45

40

60

020

30

stenosis8075

50—

76

—

- -

—

220195

205

150

250

225

250

1S5

200240

175

190

280g175

165

LVEDPt(mm. water)

C E

—

—

—

—

—

—

—

—

105

95

—

SI

—

—

63

—

70

—

—

—

—

90

SO2S

42

67

75

75

63

*Na intake, 60 niEq./day.§These values represent inferior vena caval pressure ratlier than mean atrial pressure.•f Pressures were measured immediately before sacrifice. C, control; E, experimental; LArEl~>P,

left ventricular end-diastolic pressure.$A -f- indicates presence of ascites; volume not measured but several hundred milliliters

present.

and "27.9 ± 4.0 for normal left ventricle(N = 15), (2) 29.7 ± 3.5 for right ven-tricle and 28.6 ± 3.0 for left ventricle fromdogs with cardiac failure produced by pro-gressive imlmonic stenosis (N1 = 7), and (3)25.0 for right ventricle and 29.0 for leftventricle of dogs with congestive failuresecondary to tricuspid insufficiency and pul-nionic stenosis (N = 3). The actomyosinyield was essentially the same for the rightand the left ventricles and no difference wasobserved in the actomyosin yield from normaland failing hearts. Also, no difference wasfound in the actomyosin yield from freshand frozen tissue. In addition to the measure-ments on cardiac actomyosin, the nitrogencontent of the soluble extract per unit oftotal muscle nitrogen was studied and foundto be unaltered in the failing heart.

Sedimentation Velocity Studies of Acto-myosin. Comparative data among animals

were obtained for actomyosin prepared fromthe right ventricle only unless otherwisestated. The typical sedimentation patternsfor actomyosin from normal and failingheart muscle were identical (fig. 1). Asingle sedimentation boundary was presentat low concentrations (below 3.0 rug./ml.)whereas at higher concentrations there wasevidence of polydispersity. These findings wereobtained from 6 normal dogs, 5 dogs withfailure secondary to pulnionic stenosis, 2dogs with tricuspid insufficiency, pulnionicstenosis and heart failure and from 3 animalswith chronic thoracic inferior vena cava con-striction and ascites. Xo difference was ob-served in the sedimentation of actomyosinprepared from fresh and from frozen muscle.

An abnormal sedimentation pattern forcardiac actomyosin from right ventricularmuscle was found in 2 of the dogs with car-diac failure produced by pulnionic stenosis

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CONTRACT 11iE PROTEINS IN HEART FAILURE %1

F.PIG. 1. Ultnicciitrii'iigal sedimentation patterns of native actomyosin from right ventricular

muscle (wedge cell and upper part of picture for all figures) and the same actoinyosin withadded ATP and the resultant formation of myosin (regular cell and lower picture in all butE). In E, sedimentation diagrams of 2 different concentrations (4.06 nig./ml. for wedgecell and 3.06' mg./nil. for regular cell) of actoinyosin are presented. A, B, and C are photo-graphs of uctomyosin from normal heart muscle whereas V, E, and F show typical sedimenta-tion patterns of cardiac actoinyosin from dogs with congestive failure. The protein concen-trations for A, B, C, D and /'' were 1.72, 4.85, '6.29, 2.34 and 4.14 nig./ml., respectively.Photographs were taken at 8, 10, 20, S, 12 and 32 min., respectively, after reaching a speedof 59,780 r.p.ni.

(dogs 1 and 7) and in 2 of the animals(dogs 4 and 5) with congestive failure sec-ondary to tricuspid insufficiency and pul-monic stenosis (table 1). A small portion ofthe sedimenting material had an S20W rangingfrom 5.0 to 6.7 (fig. 2 Left). This finding wasobserved in at least 2 separate cardiac acto-

inyosin preparations from each of the 4dogs. In dog 5 with tricuspid insufficiencyand pulmonic stenosis, actomyosin from theleft ventricle showed the same slow sediment-ing component.

Additional sediinentation velocity studieswere carried out on actomyosin preparations

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962 DAVIS, TRAPASSO, YANKOPOULOS

which were precipitated 2 or 3 times. Aslow component with an Ssow similar to thatdescribed for actomyosin from the 4 dogswith heart failure was observed in prepara-tions of normal cardiac actomyosin from rightventricle (fig. 2 Eight). Also, a second orthird precipitation of actomyosin from theright ventricle of dog 7 with cardiac failuresecondary to pulmonic stenosis showed agreater proportion of the sedimenting mate-rial as a slow component than was presentafter one precipitation (fig. 3 Left). Meas-urements of the pH of the actomyosin prep-arations from the right ventricle showed thatpH fell after a second or third precipitation.Adjustment of the pH to 5.7 in one-time pre-cipitated material was associated with the ap-pearance of a slow component not present inthe same actomyosiu at pH 6.7 (fig. 3 Bight).

Upon addition of ATP to actomyosin, thesedimenting material formed a single boun-dary which moved at the rate of myosin(fig. 1). A very small portion (less than 5per cent) of the material was not convertedto myosin and sedimeuted faster than niyo-siu. The response to ATP was the same formaterial from all animals including the 4dogs with the slow sedimentation component.

Cardiac actomyosin from 2 of the normaldogs and from 1 dog with cardiac failuresecondary to pulmonic stenosis was dialyzedagainst 0.1 M Na2CO3 before ultracentrifu-gation. Two components were observed inthe sedimentation pattern. One componentsedimented at a rate similar to that ofnryosin; the other componeut moved moreslowly (S2ow = 1.6-2.2). No difference in theresponse of actomyosin to dialysis againstNa2CO3 was observed for material from nor-mal and failing heart muscle. This responsewas similar to that described by Kominz10

for skeletal muscle myosiu dialyzed againstNa2CO:!.

Quantitative data on the sedimentation ofactomyosin and myosin are presented infigure 4. Each point for actomyosin andmyosin represents a separate enzyme prep-aration with the exception of a few of the

values for myosin obtained at concentrationsbelow 1.8 ing./ml. No differences are evi-dent for rates of the sedimentation of pro-teins from normal and failing hearts andfrom the hearts of dogs witli caval constric-tion. Sedimentation of actomyosin showed amarked dependence on protein concentration.Extrapolation of the sedimeutation coefficientto zero protein concentration yielded a valueof 70 to 90. The rate of movement ofmaterial formed by addition of ATP toactomyosin was similar to that described formyosin from skeletal muscle.17"10 The S2owextrapolates linearly at zero concentration toa value of 6.25 for normal material aud to sim-ilar values for the experimental material.

Viscosity Measurements. No difference inthe viscosity of actomyosin from normal andfailing cardiac muscle was detected frommeasurements at 0 or at 22.5 C. with 2 vis-cosimeters which produced widely differentmean velocity gradients (fig. 5). Also, car-diac actomj'osin from dogs with thoracic in-ferior vena cava constriction showed a nor-mal viscosity. The logarithmic plot of i;reiagainst the concentration of actomyosin showsthat the Arrhenius relation

log r)rei = K concentrationholds for cardiac actomyosin as it does forskeletal actomyosin.20 The slopes of the lines(fig. 5) were essentially the same with the 2types of viscosimeters for measurements at 0and 22.5 C. The explanation for this findingis that measurements with size I viscosimeter(high /?) were made at 0 and with size IIviscosimeter (low /?) were made at 22.5 C.Apparently, the viscosity was sufficientlygreater at low temperatures to counteract theeffect of a high mean velocity gradient andvice versa. The data indicate, therefore, adependence of viscosity on the mean velocitygradient, a finding described for rabbitskeletal muscle actomyosin.21

The viscosity response to ATP was studiedon actomyosin from 6 normal dogs and 6animals with cardiac failure (table 2). The"ATP sensitivity," which is an index of theactin content of the actomyosin, ranged from

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CONTRACTILE PROTEINS IN HEART FAILURE 963

103 to 159 per cent for the normal material.This range is comparable to that of 97 to 179per cent reported by Portzehl and co-workers14 for skeletal muscle actomyosin.Actomyosin from 5 of the 6 dogs with heartfailure showed a similar response (103 to.1.30 per cent). In the remaining animal theresponse to ATP was low, but the initialjtis-cosity number was lower than all other valuesobtained for this animal. The drop in specificviscosity per unit of actomyosin concentra-tion in response to ATP showed no differ-ence between cardiac actomyosin from nor-mal and failing hearts.

ATP-ase Activity of Actomyosin. Studies,were first conducted to determine if freezingnormal cardiac muscle before extraction ofactomyosin altered the ATP-ase activity. Xodifference in the ATP-ase activity of acto-myosin from fresh and from frozen musclewas found. Thereafter, observations on theATP-ase activity of actomyosin were made onboth fresh and frozen material. The ratesof enzymatic hydrolysis of ATP by actomyo-sin from normal and failing cardiac muscle(dogs with progressive pulmonic stenosis)were not detectably different for measure-ments made with 0.1 M Ca++. Examinationof the ATP-ase activity of cardiac acto-myosin from clogs with tricuspid insufficiencyand pulmonic stenosis was made with 0.01M Ca++ which gave greater ATP-ase activityfor the normal material than with 0.1 MCa++. With 0.0.1 M Ca++, the rate of hydroly-sis of ATP appears to be reduced for theactomyosin from failing muscle, but studieswere conducted on actomyosin from 3 dogsonly so that no definitive conclusions can bemade.

DISCUSSION

In previous studies of contractile proteinsfrom the failing myocardium,D~8 little at-tention was given to either the source of thecardiac muscle (right or left ventricle) inrelation to which ventricle was failing or therelationship of the findings for the contractileproteins to the other features characteristicof congestive heart failure. Benson1""7 found

TABLE 2.—Viscosity Response of Acto'mffosin toAdenosine Triphosphate

DOB

Xorniald89

10111213

Concentration ofactomyosin(mpr./ml.)

dogs1.12

.79

.95

.871.15

.73

.307

.254

.280

.291

.230

.248

ZTIafterATP

.151

.119

.108

.138

.097

.105

ATPsensitivity' <#>

1031121,5!)I l l1'2713C

Heart failure produced by progressive pulmonk'stenosis

4 .96 .313 .150 1090 1.11 .1.79 .102 7G8 1.12 .292 .127 130

Lluiirt fa i lure secondary to pulmonic s tenosis andti ' ieuspid insufficiency

1 1.07 .2S8 .122 13(i2 .85 .258 .11.2 1304 1.23 .350 .175 103

no difference between actomyosin extractedfrom right and left ventricles in dogs withpure right-sided cardiac failure and only 3of the 6 dogs studied had aseites at the timeof sacrifice. Olson and associates8 used theentire right and left ventricular mass as asource of muscle for extraction of myosin.The question arises as to whether the pre-viously reported findings of abnormal con-tractile proteins in heart failure""8 have sig-nificance in the pathogenesis of cardiac failure.

In this and other studies,10 observationswere made to evaluate left ventricular func-tion and to define the changes associated withcongestive failure which might conceivablyalter the myoeardiaL proteins. No evidencefor left ventricular failure was obtained fromstudies of dogs with cardiac decompensationsecondary to progressive pulmonic stenosis orto a combination of tricuspid insufficiencyand pulmonic stenosis. Left ventricular end-diastolic pressure was normal in both prep-arations. There was no evidence of left ven-tricular hypertrophy or of chronic passive con-gestion of the lungs in the dogs with tricuspidinsufficiency and pulmonic stenosis. Previousstudies11 of dogs with heart failure produced

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964 DAVIS, TRAPASSO, YANKOPOULOS

Fid. 2 Top. Sedimentation patterns for cardiac actomyosin (wedge cell) and myosin (regularcoll) from right ventricular muscle. Left, proteins from a dog with experimental heartfailure; actomyosin was precipitated one time. The abnormal slow component (SAM- = 5.91)was present in the actomyosin preparation. Bight, two-time precipitated aetomyosin fromnormal heart muscle is in the wedge cell and a slow component is present while the sameactomyosin plus ATP is in the regular cell. The protein concentrations wore 4.68 and 5.13rng./ml. for Left and Right respectively. Photographs were made at 20 min. after reachinga speed of 59,780 r.p.m. in both instances.

FiG. 3 Bottom. Effects of additional precipitation {Left) and lowering the pH {Right) on the

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CONTRACTILE PROTEINS IN HEART FAILURE 90")

by progressive puhnonic stenosis have demon-strated marked depression of right ventricu-lar function. In this series of animals withheart failure secondary to tricuspid insuffi-ciency and pulmonic stenosis, there was rightventricular hypertrophy10 and the pressurein the right atrium was consistently elevated.Barger12 demonstrated impairment of rightventricular function in heart lung prepar-ations of dogs with chronic failure secondaryto trieuspid insufficiency and puhnonic steno-sis; no measurements of left ventricular func-tion were made since filling pressures weremeasured only on the right: side of the heart.Because isolated right ventricular failure waspresent in both types of animal preparations,comparison of normal cardiac actomyosinwith material from dogs in failure was madeon actomyosin from the right ventricle only,except for studies of actomyosin yield.

No difference in. the yield of actomyosinfrom normal and failing heart muscle wasfound. This result was obtained for cardiacactomyosin from both experimental animalpreparations. The aetomyosin yield per gramof total protein is slightly lower than Ben-son7 reported. This is attributable to thepresent higher values for the total proteincontent of heart muscle. In the present study,the total protein content of muscle was de-termined by digesting 1 Gin. samples ofmuscle for Kjeldahl analysis rather than ana-lyzing an aliquot of the homogenate. In ourlaboratory, this latter procedure yielded vari-able and unreproducible results regardless ofthe completeness of homogenization.

T Jit raceri trifugal sedimentation velocitystudies of actomyosin from normal right ven-tricular muscle showed essentially the samesedimentation pattern and the same concen-tration dependence of the sedimentation

so

4 3

2 0 W

3 0

15

6

0

NORMAL

0 v

'

-

-

0

o

m

2 3 4 5 6 7 8

PROTEIN M6./ML.

I 2 3 4 5 6 7 S i

PROTEIN MG./ML.

CARDIAC FAILURE( T I • PS)

AND THORACIC IVC "CONSTRICTION

W2-1

PROTEIN MG/ML.

1 y,'t HEART FAILURE

H . 1 -

1 I t HEART tUVJK .

- .«• ( 1 1 * PS) 1

, , . NORMAL CAROUC ; , \ h «ART FAILURE

.i- NORMAL SAELCtAL ° 4 T EMP •( f ' -S*C

- . | -

PIG. 4 Top. SeclimeiitJitioii constiints for Mctoniyosin(open symbols) and niyosin (solid symbols) plottedugiiinst protein concentration. Different symbols, ;\c-tomyosiu from different dog.-*.

FIG. o Bottom. Relative viscosity (n rel) for acto-myosin plotted against protein concentration. Dif-ferent symbols, actomyosin from different animals.

constants as reported for skeletal muscleactomyosin.14 No difference was detected inthe plots of Ssow against protein concentra-tion for actomyosin from normal and failingheart muscle. The response of actomyosin toATP as determined by the sedimentation pat-tern and sedimentation constants of the re-sultant myosin was identical for all materialfrom normal and failing hearts. The sedi-mentation pattern of material obtained bydialysis of actoinyosin against NaoCO.i wasthe same for normal and failing heart muscle.

Abnormal sedimentation patterns were ob-

sedimentation of cardiac actomyosin from the right ventricle of a dog with cardiac failure.Left, actomyosin was precipitated one time (wedge cell) and three times (regular cell).Tlio quantity of the abnormal slow component increased with additional precipitation. Might,the pH of the actomyosin was 6.70 in the wedge cell and 5.78 for the same actomyosin intlio regular cell. Lowering the pH increased the amount of the slow component present. Theprotein concentrations were 5.17 and 4.90 mg./ml., respectively. Photographs were takenat 33 and 24 tnin., respectively, after reaching a speed of 59,780 r.p.m.

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966 DAVIS, TRAPASSO, YANKOPOULOS

served, however, for cardiac actomyosin from2 dogs with cardiac failure secondary to pro-gressive pulmonic stenosis and from 2 dogswith heart failure produced by tricuspid in-sufficiency and pulmonic stenosis. A smallportion of the sedimenting material movedwith a rate similar to that of myosin. Thematerial probably represents myosin whichwas uncombined with actin, or a degradationor polymerization product of myosin. Thisfinding is similar to the reported consistentobservation of uncombined myosin in acto-myosin preparations from failing heart mus-cle by Benson.7

Viscosity studies were also made on acto-myosin from 2 of the 4 dogs for which theabnormal sedimentation pattern was observed.The viscosity was not reduced, as would beexpected if actomyosin were partially frag-mented into myosin and actin. The viscosityresponse of actomyosin from these 2 dogsto ATP (ATP sensitivity) was similar tothat for normal cardiac actomyosin and forskeletal muscle actomyosin.1* These data showthat the actin content of the actomyosin Avasnot reduced. The present findings are in con-trast to those of Benson7 who reported alower ATP sensitivity for normal cardiacactomyosin than that found here and a fur-ther reduction in the ATP sensitivity of acto-myosin from failing heart muscle as com-pared with normal material. A low ATPsensitivity for actomyosin might result fromfragmentation of actomyosin into actin anduncombined myosin or from inadequate ex-traction of actin from muscle. The presentaverage value of 124 per cent for the ATPsensitivity of actomyosin from normal rightventricular muscle is considerably higherthan the value of 86.3 per cent reported byBenson for normal cardiac aetomyosin. Thedata show, therefore, that more complete ex-traction of actin was obtained in the presentpreparations than in those of Benson.

What is the significance of the slow com-ponent in sedimentation patterns of actomyo-sin from failing right ventricular muscle V Theduration and degree of cardiac failure and

the genera] condition of these 4 dogs did notdiffer from the other animals with heart fail-ure. The finding of the abnormal sedimenta-tion pattern for actomyosin from the leftventricle of one of the 4 dogs suggests thatthe slow sedimentation component is not char-acteristic of a failing myocardium since theleft ventricle was not in failure.

Several other findings suggest that theabnormal sedimentation component may bean extraction or preparation artifact. First,the relative amount of the slow componentwas increased by repeated precipitation whenthe pH was not adjusted to pll 6.8. Sec-ondly, a reduction in pll of the actomyosinpreparation increased the quantity of theslow component. Finally, the slow componentappeared in actomyosin prepared from nor-mal cardiac muscle after the second andthird precipitation in the absence of ad-justment of pH to 6.8. Therefore, these datado not support the concept that actomyosinpresent in the functioning hearts of dogswith experimental congestive failure is par-tially fragmented into uncombined myosinand that such an abnormality constitutes aprimary defect in the failing myocardium.The occurrence of one-time precipitated acto-myosin preparations with a slow sedimenta-tion component solely in the experimentalmaterial remains unexplained.

SUMMARY

Aetomyosin from cardiac muscle of dogswith heart failure secondary to progressivepulmonic stenosis or to a combination oftricuspid insufficiency and pulmonic stenosiswas characterized by ultracentrifugal sedi-mentation velocity, viscosity and ATP-asemeasurements. Cardiac actomyosin from nor-mal animals and from dogs with thoracic in-ferior vena cava constriction provided con-trol material. The sedimentation patterns andsedimentation constants for actomyosin fromthe right ventricles of normal hearts and thehearts of dogs with thoracic caval constric-tion were not detectably different from theresults reported by others for skeletal muscle

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CONTRACTILE PROTEINS IN HEART FAILURE 967

actomyosin. Seven of the dogs with cardiacfailure showed normal sedimentation pat-terns and constants for cardiac actomyosinfrom right ventricular muscle. In the other 4dogs with cardiac failure, a small portionof the sediinenting protein had an Sow rang-ing from 5.0 to 6.7. Additional precipitationor reduction in the pH of the actomyosinincreased the relative amount of this slowcomponent. Also, repeated precipitation ofactomyosin from normal dogs without ad-justment of the pH to 6.8 yielded a similarslow component in the sedimentation pattern.Cardiac aetomyosiu from all normal and ex-perimental animals responded to ATP byformation of myosin; the S2ow at zero con-centration was 6.25, a value similar to thatreported for skeletal muscle myosin. Theviscosity of actomyosin and the viscosity re-sponse to ATP were the same for material fromnormal dogs and dogs with experimentalfailure; also, the present viscosity data oncardiac actomyosin are similar to those re-ported for skeletal muscle actomyosin. Nodifference was observed in the ATP-ase ac-tivity of normal and experimental material.It seems likely that the slow componentpresent in the sedimentation patterns ofactomyosin from the 4 dogs with heart failurereflects an extraction or preparation artifact.It is concluded that these data do not sup-port the concept that the contractile proteinsare altered in experimental heart failure.

ACKNOWLEDGMENTS

We are indebted to Doctors W. R. Carroll, E.Mihalyi, K. Laki, W. J. Bowen and D. R.Kominz for helpful advice and criticism. Dawn B.Willis and Harry K. Marshall rendered valuabletechnical assistance.

SUMMAKIO JN I N T E R L I N G U A

Le actomyosina ab musculo cardiac decanes in disfallimento cardiac secundari a pro-gressive stenosis pulmonic 0 a un combina-tion de insufficientia tricuspidic con stenosispulmonic esseva characterisate per mesura-tiones del velocitate de sedimentation perultracentrifugation, del viscositate, e de ad-

enosino-triphosphatase. Le actomyosina car-diac ab canes normal e ab canes con con-striction del vena cave inferior in le thoraceprovideva le valores de controlo. Le eonfigura-tiones de sedimentation e le constantes desedimentation pro actomyosina ab le ven-triculo dextere de cordes normal e ab lecorde de canes con constriction del vena cavethoracic non differeva detegibilemente ab leresultatos reportate per alteres pro actomyo-sina ab musculo skeletic. Septe del canes condisfallimento cardiac exhibiva normal con-figurationes e constantes de sedimentation proactomyosina ab musculo dextero-ventricular.In le altere 4 canes con disfallimento cardiac,un micre portion del proteina sedimentatehabeva un S20W in le intervallo ab 5,0 a 6,7.Precipitation additional 0 reduction in le pHdel actomyosina augmentava le quautitate re-lative de iste lente componente. In plus, lerepetite precipitation de actomyosina abcanes normal, sin rectification del pH a 6,8,produceva un simile lente componente in leconfiguration sedimentatori. Actomyosina car-diac ab omne le animates normal e experi-mental respondeva a adenosino-triphosphatoper le formation de myosina. Le SL>O»- al con-centration zero esseva 6,25, un valor similea illo reportate pro myosina de musculoskeletic. Le viscositate de actomyosina e leresponsa de iste viscositate a adenosino-tri-phosphato esseva le mesme pro material abcanes normal como pro material ab canescon disfallimento cardiac de origiue experi-mental. In plus, le prescnte datos pro le vis-cositate de actomyosina cardiac es simile aldatos reportate pro actomyosina de musculoskeletic. Nulle differentia esseva observate inle activitate de adenosino-triphosphatase in-ter material normal e material experimental.II pare probabile que le lente componeute inle configuration del sedimentation de acto-myosina ab le 4 canes con disfallimento car-diac reflecte un artefacto de extraction 0preparation. Es concludite que le presentedatos non supporta le conception que le pro-teinas contractil es alterate in disfallimentocardiac de origine experimental.

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968 DAVIS, TRAPASSO, YANKOPOULOS

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CASPERJAMES O. DAVIS, MARY TRAPASSO, NICHOLAS A. YANKOPOULOS and ALFRED

FailureStudies of Actomyosin from Cardiac Muscle of Dogs with Experimental Congestive Heart

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1959 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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