High Phosphate Compounds Myocardium Experimental ... · High energy phosphate compounds are...

Journal of Clinical InvestigationVol. 44, No. 2, 1965

High Energy Phosphate Compounds in the Myocardiumduring Experimental Congestive Heart Failure.Purine and Pyrimidine Nucleotides, Creatine,

and Creatine Phosphate in Normal andin Failing Hearts *

ARTHURC. Fox, NORMANS. WIKLER, AND GEORGEE. REED

(From the Departments of Medicine and Surgery, New York University School of Medicine,New York, N. Y.)

High energy phosphate compounds are gen-erally considered essential for muscular contrac-tion although it is not clear how their chemicalenergy is ultimately made available for trans-formation into mechanical work (1-3). Accord-ing to classical concepts, hydrolysis of adenosinetriphosphate (ATP) by myofibrillar .ATPase(adenosine triphosphatase) occurs at some timeduring repetitive cycles of muscular contractionand relaxation, and as a result adenosine diphos-phate (ADP) is produced. Delivery of this ADPto mitochondria for regeneration into ATP canserve as an important regulator of the activityof mitochondrial oxidative phosphorylation (4,5). In failing hearts, changes in oxidative phos-phorylation or its products might accordingly re-flect primary changes at the mitochondria oraltered delivery of ADP from the failing myo-fibrils (6).

Many studies of normal and failing hearts haveexamined the processes involved in the formationand utilization of high energy phosphate com-pounds (7). During spontaneous heart failurein man (8-10) and induced heart failure in dogs(11), there were no significant changes in uptakeby the myocardium of oxygen or of the diversesubstrates from which chemical energy is liberatedand then conserved as nucleoside triphosphatesor as creatine phosphate (CrP). However, small,long-term, or localized alterations in uptake ofexogenous substrates might not have been de-tected by acute measurements of arteriovenous

* Submitted for publication December 19, 1963; ac-cepted October 15, 1964.

Supported by grant H-3312 from the National Institutesof Health, U. S. Public Health Service.

differences across intact hearts in situ (12).Concentrations of high energy phosphates havebeen reported to be normal in isolated, acutelyfailing dog hearts, guinea pig atria, and cat papil-lary muscles (13-15) and in chronically failinghearts from dogs with tricuspid insufficiency andpulmonary stenosis or with aortic insufficiency( 11, 16). Muscle preparations from failing canineand human hearts have shown decreased con-tractility (17, 18) and decreased ATPase activity(19), but myofibrillar proteins from failing caninehearts have been reported to be both normal (20)and altered (21).

Recently, changes have been observed in themorphology of mitochondria from failing heartsof dogs with aortic stenosis (22) and in themetabolic responses of mitochondria from failinghearts of guinea pigs with aortic stenosis (23,24). Improved methods for isolation, separation,and specific assay of high energy phosphate com-pounds have made possible more precise measure-ments of tissue levels of these labile compounds(25-27). With the use of such improved tech-niques, a recent study found significant decreasesin levels of ATP, CrP, and creatine (Cr) infailing hearts from guinea pigs with aortic steno-sis (28). Since the status of labile phosphatecompounds in the chronically failing heart didnot yet seem settled, it was considered pertinentto re-examine the content of purine and pyrimi-dine nucleotides and CrP in hearts from dogswith unequivocally severe, chronic, low outputcardiac failure.

This report. deals with the production of chroniccongestive heart failure in dogs and with- meas-urement of the levels -of purine and pyrimidine

2u2

HIGH ENERGYPHOSPHATESDURING HEARTFAILURE

nucleotides, Cr, and CrP in the right and leftventricles from the hearts of these animals. Therewere significant decreases in concentrations ofATP, Cr, and CrP in severely stressed rightventricles from hearts in chronic failure due topulmonary arterial stenosis.1

Methods

Production of congestive heart failure in dogs. Twomethods that have been widely used for the productionof chronic congestive heart failure in dogs were exam-

ined further. Fourteen dogs were subjected to a com-

bination of tricuspid insufficiency and pulmonary arterialstenosis (29). Severe tricuspid insufficiency was pro-

duced by avulsion of all tricuspid chordae tendineae ex-

cept those of the septal leaflet; 2 weeks later, as muchtransverse area of the main pulmonary artery was re-

sected as the animal could tolerate (usually 70%). In 17animals, isolated, severe stenosis of the main pulmonaryartery was successfully produced by the method of Davis,Hyatt, and Howell (30). Ligatures placed around themain pulmonary artery were progressively tightened atweekly intervals to maintain right ventricular systolicpressures of at least 75 to 100 mmHg. When breathless-ness, ascites, edema, and engorgement of the liver ap-

peared, chest X rays were taken to demonstrate cardiacenlargement, and hemodynamic studies were performedby right heart catheterization. A basal state was as-

sured by light thiopental anesthesia, and adequate spon-

taneous oxygenation was maintained through a cuffedendotracheal tube. The hemodynamic criteria for heartfailure included the presence of marked elevations ofright ventricular end diastolic pressure, measured at thetrough of the transmitted a wave, and significant in-creases in a-v 02 difference. Animals with evidence ofsevere anemia, malnutrition, infection, or pulmonary in-farction were discarded.

Right ventricular hypertrophy without congestive heartfailure was produced with pulmonary arterial ligaturesthat were tightened sufficiently to cause sustained ele-vations in right ventricular systolic pressures without in-

creases in the end diastolic pressures or in a-v 02

differences.

1 The principal abbreviations used in this report are as

follows: AMP, ADP, ATP, GMP, GDP, GTP, UMP,UDP, UTP= the 5' mono-, di-, and triphosphates ofadenosine, guanosine, and uridine; CMP, CTP=the 5'mono- and triphosphates of cytidine; IMP = the 5' mono-

phosphate of inosine; NAD= nicotinamide adenine dinu-cleotide (DPN); FAD= flavin-adenine dinucleotide;Cr = creatine; CrP = creatine phosphate; Pi = inorganicphosphate; TASP= total acid-soluble phosphate; A260 =

absorbance of ultraviolet (UV) light at 260 miu; a-v

02 difference = the difference in oxygen content betweenfemoral arterial and mixed venous blood from the rightventricle or the pulmonary artery.

Rapid extirpation of normal, hypertrophied, or failinghearts was effected while the hearts were beating or im-mediately after induction of cardiac arrest. The chestswere opened during gentle, assisted respiration with oxy-gen, and pressures in the right and left ventricles of thefailing hearts were measured by direct puncture of thetwo chambers. Arrest was produced by rapid injectioninto the cross-clamped aorta of 5 ml of 5% potassiumcitrate at pH 9 (31). Fewer than 3 to 5 QRScomplexesoccurred between the beginning of the injection and totalarrest. The cardiac apex was grasped with a sharp towelclip, the atria and great vessels were quickly severed, andthe intact ventricles were plunged into a Dewar flaskfilled with liquid nitrogen. The procedure rarely ex-ceeded 6 seconds; specimens were discarded if extirpationand freezing took more than 10 seconds. In five stud-ies, the quadriceps femoris muscle was exposed withoutdamage to its blood supply and was rapidly excisedand frozen as the aorta was clamped or as the beatingheart was excised.

Acid-soluble extracts of the muscle specimens wereprepared in a cold room at 40 C. The free walls of bothventricles were rapidly separated and fragmented. Thefrozen fragments were chipped clean of visible bloodvessels, visceral pericardium, fat, fibrous tissue, and ad-herent blood, and pulverized to a fine powder underliquid nitrogen in a large porcelain mortar. The pow-dered muscle was rapidly mixed into cold, 0/6 N per-chloric acid, and homogenized at 45,000 rpm for 15 min-utes with a VirTis model 45 tissue homogenizer. Theacid extract was separated in a centrifuge at 40 C and25,000 g. The clear supernatant fluid was combined withcentrifuged water washings of the precipitate and neutra-lized in an ice bath with 5 N KOHto pH 7.0. Partialfreezing precipitated KC104, which was removed by fil-tration. The volume of the filtrate was measured and itscontent of material absorbing at 260 mA (A260) deter-mined; then it was stored at -200 C until analyzed.

Fat-free, dry weights were determined for each musclesample to correct for variations in water and fat con-tent (32). Since significant increases in collagen con-tent had not been found in human hearts failing from awide variety of causes (33), corrections for collagen con-tent were not made.

Separation and measurement of the nucleotides in ex-tracts of normal and failing hearts were achieved by gradi-ent elution from Dowex 1 (Cl) X-10 resin columns(25), which were 20 X 1.25 cm in size. Between 90%oand 95%o of the A260 material in the extracts was regu-larly absorbed by the resin. Compressed air was usedto force eluting solutions of increasing concentrations offormic acid and ammonium formate through a mixingflask and then through the columns, which were encasedin a tap water jacket. Serial 5-ml fractions were col-lected by a Technicon fraction collector with a drop-counting attachment, and the A260 of each fraction wasread in a Beckman DU spectrophotometer. The eluatescomprising each significant peak of UV absorbancy weredried on a VirTis freeze-dryer to remove eluting salts

203

ARTHURC. FOX, NORMANS. WIKLER, AND GEORGEE. REED

that interfered with accurate spectrophotometric analy-sis. The dried nucleotide powders were dissolved inknown volumes of water, identified by their absorptionspectra at pH 1, and quantitated by measurement of ab-sorbancy at their absorption maxima and by applicationof standard molar absorbancy indexes. Precise identi-fication of NAD was obtained by demonstration of itsUV spectrum shift with cyanide, and of FAD by its mi-gration and fluorescence in paper chromatograms. Fur-ther separation of compounds that were not well iso-lated by the formic acid system was effected by Phase IIchromatography on Dowex 1 resin columns 8 X 1 cm insize. Eluates were obtained with concentration gradientsof ammonium formate buffer at pH 5.

The technique was standardized by elution from theresin columns of pure nucleotides mixed in amountsanticipated for 25 g of heart muscle. Average recoveryrates of significant compounds were as follows: ATP,98%o; ADP, 95%; NAD, 87%; IMP, 96%, CTP, 88%;CMP, 94%. Recovery rates of several compounds wereartificially increased by breakdown of the more concen-trated and labile nucleoside triphosphates: AMP, 111%;UMP, 122%; GDP, 112%. UDPwas variably recoveredat about 50%. Measurements of nucleotides in dupli-cate specimens from the same ventricle yielded valuesthat agreed within 5%. Because it was difficult to re-move completely the concentrated ammonium formatesalts added during Phase II separation of UTP andGTP, recoveries were estimated directly from the UTPand GTP peaks in the Phase II chromatograms. Sincethese recovery rates were only 50%, the values subse-quently found for UTP and GTP in tissues weredoubled to give a better approximation of in vivo levels.The identity of each compound isolated from the columnsin the early part of the study was confirmed by paperchromatography in two directions (34). Quantitative elu-tions from paper of the principal separated nucleotidesdid not yield recoveries equal to those obtained with theresin-spectrophotometric system.

Total acid-soluble phosphate was measured in sam-ples of the muscle extracts after all bound phosphateswere hydrolyzed by 5 N H2SO4 in an oil bath heatedat 1600 C for 1 hour. The liberated phosphate wasmeasured (35) with standard recovery rates of 95%.This procedure was also used early in the study to con-firm the spectrophotometric quantitation of eluted, driednucleotides; the results obtained by the two methodschecked regularly within 5%. Analysis of CrP, ATP,and ADP by measurement of the phosphate liberated byselective hydrolysis of CrP and "acid labile" nucleotidephosphates ("p-p") (35) did not prove as reliable asthe ion exchange method.

Creatine phosphate was measured by two methods.The method of Furchgott and De Gubareff (36) de-pends upon measurement of free inorganic phosphateand then of additional phosphate liberated by carefullycontrolled acid hydrolysis of CrP. The- method of Ennorand Rosenberg (37) measures the colored diacetyl com-pound formed first with free creatine base and then with

the additional creatine base liberated by acid hydrolysisof CrP. These procedures also yielded values for in-organic phosphate and for free and total creatine. Themethods were standardized by analysis of known mixturesof Cr, CrP, and Pi and were found to be accurate within5%. Addition of contaminating ATP did not affect theaccuracy of the standard measurements.

Results

Evaluation of the severity of congestive heartfailure

Tricuspid insufficiency and pulmonary arterialstenosis, as combined lesions, produced ascites inonly six of 14 animals despite frequent exerciseon a treadmill, and animals with severe ascitescould exercise well. Spontaneous diureses oc-curred frequently, with abrupt disappearance ofascites. Even when severe circulatory congestionhad been present for periods of from 3 to 10months, mean right atrial pressures ranged from11 to 15 mmHg, right ventricular systolic pres-sures were only 22 to 45 mmHg, and rightventricular end diastolic pressures were from 4to 10 mmHg. Values for a-v 02 differencesnever exceeded 6 vol per 100 ml.

Progressive pulmonary arterial stenosis was amore reliable method for producing severe con-gestive heart failure. Many animals with tight-ened ligatures died in failure before studies couldbe completed. Systolic pressures in the rightventricle had been markedly elevated for as longas 6 weeks before signs of congestive heart failureappeared. End diastolic pressures were seldomelevated before the appearance of severe ascites,hepatic engorgement, leg edema, and pleural ef-fusions. Breathlessness at rest and significantincreases in a-v 02 difference were the lastchanges to appear. When there was severe con-gestive heart failure, pressure gradients acrossthe stenosed pulmonary arteries remained high,although systolic pressures in the right ventricleshad fallen by as much as 30% from peak levels.Ventricular alternans was sometimes present.The contours of the right atrial pressure curvesdid not reflect the severe degree of tricuspid re-gurgitation apparent in exposed hearts. Theright ventricles were markedly dilated and hyper-trophied. The oxygen contents and saturationsof femoral arterial blood indicated that althoughsome animals were mildly anemic, all were well

204

HIGH ENERGYPHOSPHATESDURINGHEARTFAILURE 205

TABLE I

Hemodynamic measurements

Intracardiac pressures Blood gases

Days By catheter By direct measurementwith Arterial Mixed

pulmo- Right (open chest) oxygen Arterial venous a-v 02Animal nary atrium Right Right Left satura- oxygen oxygen differ-

no. stenosis (mean) ventricle ventricle ventricle tion content content ence

mmHg mmHg mmHg mmHg % uol/100 ml vol/100 ml vol/100 ml

A. Animals with severe heart failure produced by pulmonary arterial ligatures

71 62 941 17 9 95 17.29 7.94 9.35

-3,14 8,14 8,11

74 70 1172 57 13 96 16.5 9.6 7.2

0,18 6,17 4,7

64 110, 57 1203 74 19 13.7 6.42 6.65

0,11 10,17 6,12

77 734 60 15 100 16.15 5.36 10.97

-5,15 1,11

96 925 80 11 101 19.44 11.97 7.47

8,14 7,14

79 100 1006 36 13 102 20.67 10.82 9.85

0,13 3,8 1,6

58 53 1057 19 16 15.50 8.54 6.96

10,20 1, 13 -4, 7

99 1138 90 15 20.14 10.13 9.83

4,22 8,25

96 52 689 40 11 102 16.39 8.36 8.03

-2,13 2,7 -1,2

97 64 9110* 39 11 100 13.91 6.91 6.95

2,17 5,13 3,6

75 75 10011 41 16 105 12.63 5.62 7.01

-4,14 0,14 0,4

75 95 89lla* 100 15 99 20.44 8.26 12.2

2,18 5,25 0,4

65 53 7012 34 11 99 17.79 11.01 6.8

1,2 2,7 0,4

53,68 68.89 110,11213 29 10 102 15.50 7.97 7.53

5,10 12,12 3,4

40 60 7514 37 14 100 9.97 2.24 7.73

-2,14 5,12 1,3

8715 36 15 94 14.7 5.8 8.9

7, 11

6816 34 14 103 16.63 10.01 6.62

0,17

7217 40 14 100 15.23 9.2 6.0

2,16

76 77 95Average 48 13 100 16.3 8.1 8.2

2,14 5,14 2,6


TABLE i-Continued

Intracardiac pressures Blood gases

Days By catheter By direct measurementwith Arterial Mixed

pulmo- Right (open chest) oxygen Arterial venous a-v 02Animal nary atrium Right Right Left satura- oxygen oxygen differ-

no. stenosis (mean) ventricle ventricle ventricle tion content content ence

mmHg mmHg mmHg mmHg % vol/100 ml vol/100 ml vol/100 ml

B. Animals with pulmonary arterial ligatures and chronic right ventricular hypertrophy without heart failure

521 170 0 104 22:94 20.06 2.28

-5,0

782 40 0 105 17.44 13.03 4.41

-4, 3

50, 75a 43 -1 107 19.53 15.37 4.16

-10, -1

804 39 0 108 28.01 24.79 3.22

-10, 0

505 59 103 20.70 16.23 4.47

-5,3

586 69 -2 107 20.53 15.63 4.90

-12,0

66Average 70 0 105 21.5 17.5 4.0N-8, 1

* Not examined chemically.

oxygenated. With thoracotomy, systolic pres-sures in both ventricles fell slightly (38); enddiastolic pressures were uniformly high in theright ventricles, but remained normal or wereonly slightly elevated in the left ventricles. Hearts10 and 1lA were discarded because their ex-tirpation was too slow (Table I, A).

Right ventricular hypertrophy was evident inthe chest X rays and exposed hearts of six ani-mals in which a constant degree of pulmonaryarterial stenosis and right ventricular systolichypertension had been present for long periods.No clinical or hemodynamic evidence for heartfailure was present in these animals. The needfor expeditious handling precluded weighing orprecise measurement of the hearts, but the thick-ness of the right ventricular walls, estimated bygross inspection of the frozen, transected speci-mens, certainly equaled that present in the fail-ing hearts. Arterial oxygen saturations werehigh in this group, as they often were in thefailure and control groups (Table I, B).

Nucleotide content

Qualitative. The qualitative patterns found forright and left ventricles from each normal and

failing heart were identical; no analyses for nu-cleotides were made in the hypertrophied controlgroups. Compounds identified by ion exchangechromatography, UV absorption spectra, andpaper chromatography included NAD, CMP,AMP, IMP, UMP, FAD, UDP, GDP, CTP,ATP, UTP, and GTP (Figure 1). The small"ADPx" peak that tailed after the principal ADPpeak was shown by its UV spectrum and Rfvalue to consist primarily of ADP and was in-cluded in the quantitative values for ADP. Ade-quate resolution of the IMP/UMP peak wasrarely possible, and much of this material prob-ably represented degradation products from othernucleotides.

The 5 to 10% of A260 material in the acid-soluble extracts that did not adsorb on the resincolumns was not clearly defined. Paper chroma-tography of this fraction showed that it did notcontain nucleotides; its elution characteristics andabsorption spectrum resembled those of inosine.

A normal human myocardium yielded a nu-cleotide pattern qualitatively similar to that shownfor the canine right ventricle in Figure 1. Fif-teen minutes after ventricular fibrillation had beeninduced by penal electrocution of a 30-year-old

206

HIGH ENERGYPHOSPHATESDURINGHEARTFAILURE

healthy man, the anatomically normal heart wasremoved and frozen in liquid nitrogen. Despiteconsiderable hydrolysis of the labile nucleosidetriphosphates during the delay in tissue sampling,the following compounds were clearly identified inright and left ventricles: inosine, CMP, NAD,AMP, UMP, ADP, GDP, ATP, UTP, andGTP.

Quantitative. The total amount of A260 ma-terial in the neutralized acid-soluble extracts ofnormal and failing hearts was variable and nota reliable index of nucleotide content. Of thetotal nucleoside triphosphates in normal ventri-cles, ATP comprised 92 to 94%, reflecting theimportance of this compound in myocardial en-ergy metabolism; CTP, UTP, and GTP com-prised the remaining 6 to 8% (Table II). Of

4.424.49 A 4.35

3.6- 400 4.05

3.4-

3.2-

3.0-

2I8|

2.6

2.4-

2.2-

cc 2ao IO0N 4.0ON 0.2 MA.FwL. FA. FA. 4.0ONF.A0- 1.80

.D1.6-I

the total adenine nucleotides, ATP comprised84% in both ventricles. There were no signifi-cant differences in nucleotide content betweennormal left and right ventricles, whether thevalues found were compared statistically as pairsor as groups.

Among the eight failing hearts, the ratio be-tween wet and dry tissue weights was increasedby an average of 3%o for the left ventricles andby 5%7o for the right ventricles, reflecting a slightlygreater water content. Of the total adenine nu-cleotides, the average proportion of ATP re-mained at the normal level of 84%o in each ven-tricle. In seven of the eight failing hearts, how-ever, the concentrations of ATP in the rightventricles were from 1 to 14%o lower than inthe paired left ventricles (t = 2.76 and p =

1 200

TUBE NUMBER300 400

FIG. 1. NORMALPHASE I CHROMATOGRAM.Pattern of elution from an ion exchange columnof nucleotides present in an acid-soluble extract of a 28-g sample of normal canine right ven-tricle. Eluting solutions of formic acid (F.A.) and ammonium formate (A.F.) were addedin gradated concentrations at the arrows. A260 = absorbance of ultraviolet light at 260 miu.Other abbreviations refer to the mono-, di-, or triphosphates of adenosine, inosine, uridine,guanosine, or cytidine.

207


(6 C5 6 CS

qE0~ 0C40

00 0 0

4 CS Go m

00 _ a

00 00- '0

t- 0'0 C-

Cs00 t-'0 4

ONOO

'00_-.

.4 . co .

uo 0 r 0 '0

'C

00 _O

4 O 0 4 N

00 00 0

)o~

.0 0 0 _ 0

00 0000

00C 0eC 1 _

'e- C-Ut-. 00

*'0 e''0 ~-0 0

C-. 0 C-UN

'.0 0 \00 000 -

00 S

- 00 Z

208

._*

'C.0

00

*4)

bo

4)

04P

z

0P

H

O

4),

Ha

0

u

z

04

H

H

4.7

0 O

30.

0

n -!

C 0' 0

1-0 V) t0

CC) C00CC)

44 0~v)9 -U C-U14

CS N

0'

C0 0

00 eCo0eCl) +

04 CC)

C4

V -I

aw-t

C-)

co0CU

tk-

O :

0>

6 C,00~

c0

C-B0!

>ZU* -F--44


< 0.04). When compared as a group with thenormal hearts, the failing hearts showed decreasesin ATP concentration of 12% in the right ven-tricles (p = < 0.001) and of 6% in the left ven-tricles (p = < 0.03). Artifactual hydrolysis ofATP during extirpation of the failing hearts wasnot a likely cause for these decreases, since therewere no significant differences in the inosine-likeeffluents from the columns or in the concentra-tions of AMPand ADP. Although levels foundfor UTP, GTP, and GDPwere variably elevatedin the failing hearts, the technical limitations inthe measurement of these nucleotides made suchchanges insignificant.

The nucleotide levels in normal or failing heartsremoved after induction of cardiac arrest re-sembled those found in comparable beating hearts,although the numbers were too small for statis-tical comparison (Table II). One of the threenormal hearts removed after induced arrest wasfrom an animal in which an untightened pulmo-nary arterial ligature had been in place for 3weeks. The pressure in the right ventricle ofthis animal was 20/0 mmHg, and the presenceof the ligature did not affect the content of ATP.

Total creatine, free creatine, CrP, Pi, and TASP

Values found for CrP by the two methods ofanalysis usually agreed well although the phos-phate method gave higher results.

Normal hearts, removed while beating or inarrest. The mean concentrations of CrP andTASP in the right ventricles were higher thanin the left ventricles by 30 to 45%. The p valuefor the significance of the differences betweenpaired right and left ventricles was < 0.05. Thepercentage of total creatine present as CrP washigher in the right ventricles, but the differencebetween paired values was significant only in thebeating hearts. Induction of cardiac arrest ineither normal or failing hearts increased thepercentage of total creatine present as CrP, areflection of decreased hydrolysis of CrP (TableIII; Table IV and Figure 2).

Hypertrophied hearts, removed in arrest. Themean content of CrP in the right ventricles wasagain higher than in the left ventricles, but thedifference between paired values was not sig-nificant for this smaller group of hearts. Greater

crPTOTAL Cr

60-

50

z

aL

40

30

20

10

CARDIACMUSCLE

LVw RV

14

4

E1ilWL CHF

(BEATING)

f BN'L HYP CHF

(ARRESTED)

I7SKELETALMUSCLE

IVC LIG N'L. CHf

FIG. 2. PERCENTAGESOF TOTAL CREATINE PRESENT ASCREATINE PHOSPHATEIN CARDIAC AND SKELETAL MUSCLEFROMVARIOUS PREPARATIONS. The values for CrP rep-resent averages derived from separate measurements ofthe creatine base and inorganic phosphate liberated bycontrolled hydrolysis of heart extracts. The numbersabove the columns indicate the number of samples stud-ied. N'l = normal; Hyp = right ventricular hypertrophy;CHF= congestive heart failure; IVC Lig. = inferiorvena caval ligature.

relative hydrolysis of CrP in the left ventricleswas suggested by the higher values found forfree Cr and Pi. However, the percentage oftotal creatine present as CrP was not significantlylower in the left ventricles (Table IV, B, andFigure 2).

Normal and hypertrophied hearts removed inarrest differed significantly only in content of Piand TASP in the left ventricles (Table IV andFigure 2).

Failing hearts removed while beating. The leftventricles in this group, when compared withnormal, beating left ventricles, showed significantdecreases in total and free creatine (- 24%o), inCrP (- 29% and - 23% by the two methodsof analysis), and in Pi (- 15%o). The percent-age of total Cr present as CrP remained thesame as in the left ventricles from normal hearts.

The right ventricles showed significant de-creases from the normal in total creatine(-39%o), in free creatine (-33%o), in CrP(-53%o), and in TASP (-23%o). The per-centage of total creatine present as CrP decreasedand reached the levels found for the paired leftventricles in each heart (Table III and Figure 2).

A heart removed while beating from an animalwith mild congestive heart failure due to markedtricuspid insufficiency and pulmonary stenosisdid not show decreases in content of total creatine

209


W014m%0W-00%0 00L--X- 0\C) C-

4+eN0000-00N1C- em m m CV)mId4m 0

C-4("4C,4m m -It-t- I0%0000 to%

CV)

CV)

CI

C-IIcV)

V"

U)

t) o1v0'I o toUl ) \0 C1C -,-4

0

z2.

a-..t- \0CeW\000t-0

-U) 00 CN00%\ 0 -

#)4O'NIC4 C CNI C' CV)

+F

a#)~)CV)00%~-40%-C -1

00

--%0 000to

4+

(N esn s 0\ 0\

00 o-o

CI en 00 m- -! - -

00 0 .d CI4CI4 mV CN en)

mCV) 0 toV- -" CN -4

U)

C-.

C-I

1.4

dq

EdU b0

X-o eq I l

U40-

U)

O'00 \00%-u

\-'0 CN -H

oo osu

)

+

U) No X- 00

Ca X Ca

4.() zzz w

210

*

a)

u

._

0

4-

P

.Cd

OCd

wCl)

0a)

v

dF

a).4-

CdA

.Z Cd-

;|0>,-.a

C)

Ca4

O

_~a

C.)<

C..c'*

r20

C.)

000VC)

00

0

o.

V

u) V

Co

_ 0& 0

0

Ca

In 0

L.-

C3Ca

0

a)

a)

U)

Cd

a)

C-0

0

(a)

0

0

0.

0

0

CI00

0

CV)

0%

00

C-

00

* -U) 0

C-

Ca0

Ca'

C._

CI

U)

~0

CI

0

C-

W

*0

'0

n

4)Ca

-

a)

a)

U)C

0

Ca

bO

0.l)U)

0-

Cd Cf s

C-

Calo

,,Caa)

U.).

'Cda

a)U,t4.) b..i-Cd) U) vb

d o °

> -* 4-a)

oa E aC- aooC-

C-s* Ua)

UCa)C.)U)

U) a

Ct->: C-a) C-


U)in 0 C t- 00 0C '0 ,, 00 M 0t-oU00%OC t- 0% 0% CM 00o'd 0,- ---- -4 ' CNCMC CN CM4

0%s 00 0 t- 00 t-. CM4 00 000%N0 U) CM 0CM -4 m).4C CM4C- -- 4 CM4 CM - CM4 CM,

..4 U-) 00 00 0 CM4 CM4 .d d4 \0 \0 00 m M"4 -.4 -It 44 t- \0 't U) U) U') U') U) kl to

4M--4 '0d4 0s 0 -d'00 M - d00'0z-0 000 00\000ot

MO " C% 0%O mCM in0CM-4 U1

0

U)t 00\0 00 V-4t- t-CM 00 00 M 00

-~~~~ ~ ~ ~ ~ ~ ~ ~~~~-

C ,4c 00 00 00 00% 0%0s

MCM4UCMM" CM-41CM

s'U

U)¢ U)e U)U) +

40o000 %

CM

CM!

CM C

_ CMU)

0C 'o U)o0 U)0 C1-0000CM4 CM CM4 - CM4 CM4

U) m t- CM Z 0" CM4 W-4 CM4 v- CM

0CN CM4 U) 00U') 44 U) .d4 U) U)

U) U) -U) 0

U)m

U) .4 U U)

WU)U0CM4

C,m0%-4

'IO

o~

U)

Ul)

'0 CN0%C 00 -4 000 CO 0eZ- %r rOt-A X 0r

Ut

00_I

'0

CM

0CMO

.04\0 C14

em)

U)0 %00U)- UZ- 4'ro -'-4

00 'oC- No 0 k- 09 DO 2\0%0C- 00

t--(=)co 0 \ L 0 +I4-4 C ) n.o--OONEO \2<C-gM 4C14- -4 M-_I_ _4 -- _4 _" _- _ _- _4 _" 11 "++I-

CM!

0

4)00 0CM00CM q* tU0 0-

0

*M 00 U)C--U)C- '0 0

+ C C C M4

00

U) +4U)00 U)tin .d --tm0 U:

00.-4 0%0%00+ 0

CMU)U) )4

00 U)i- CA 0 00

CM 00 vt 0%

0 m 14Uos1 of

C4U)

0

000(Ds'0.

CM Ul) 0CM4 CM4 CM

000C~ 'U) CM4 U

0% tl4. 0%CM4 CM CM4

U') in ")

..d4

00I- "ICt--

soomoo on

- C CM CM CM

U)

+ U)U)U) U)

)00CM 00CMOCDC00C--00 C--00 00 0

"V4

Cd~~~~C

° - C '0t-'000'00% --00

0-N- 0

C4M U4 CM 4 U)e

0% 0% +4 + '0C

CM -00

0 eU00

0%

00_CM4

CM!CM CM4

r

C-- -4CM1

0\m00 \0 0NO \ C0

0 U)00'0'~l 00 '000 0\- H

-

Tl

=Lz [.z>s2'.zX eW

tCI UUUUg= == t

211

*

4)

._

4)

C:

4i

CoH

4)

.Cc4-) C-.-

0 C4-

4)4*

Ca 0%Q

4)

00

4)C

CaA

0(

U4)

Cd

4) Y0c44

X0>.

4)

(.-d

L0o

.44

4.)

0B.s

ffi b4t

-4Xq0

t.4o

1.X

ARTHURC. FOX, NORMANS. WIKLER, AND GEORGEE. REED.

00~~~vv

o 0

< t ° °V o° °84m°~~"tc

w C;eC.06

.S

e |Cl) 0S 0S t: \os

, Z V V m+060

4)4ON-,

@.X. 0 NO)W

Z eV C f)|

>. N-X_

cjU2S V V Ne 42s 0 -

S| V- |

Cd C,

r~~~~ ~ ~~~ 0-..

WV ° 0XX eeW ob

~cco zs~~~~~~~. EO00r

XQVz V V > ON040.°&)

.4 1 i E3 V

0v4L

.. .J. .4boH .4.4VJ0.O %C0O U*

oa4- U

> Cd~~~~l

CD ~~~~ci) Q O O i44 - 0 0 C

L)=.a I..-0~~~~~~~~~~~~~-4

06 09

w co

Cl'~00 fC00 ,) - 0V V +J)

0 M 00 r- O00~0~

4)~~~~)C

-~~~~~~~~~Ucd c

-~~~~ll -~ U *. p-

212

I


or CrP as large as those usual in hearts withsevere failure due to isolated pulmonary stenosis(Table III).

Failing hearts removed in arrest. The leftventricles in this group showed significant de-creases from the normal arrested hearts in totalcreatine (- 24%), in free creatine (- 23%), inCrP measured by liberated base (- 25%o), andan increase in Pi(+ 20%).

The right ventricles again showed large de-creases in total creatine (- 37%), free creatine(-33%), CrP (-43% and -33%o by thetwo methods of analysis), and in TASP(- 13%). The percentage of total creatinepresent as CrP remained the same as in arrestednormal hearts, and this value remained higherfor each right ventricle than for the correspond-ing left ventricle.

The differences between the failing and hyper-trophied hearts were substantially the same asthose between the failing and normal hearts(Table IV and Figure 2).

Partial ligation of the inferior vena cava abovethe diaphragm in three dogs produced massiveascites, hindquarter edema, liver congestion, andtissue wasting like that seen in the animalswith congestive heart failure. Intracardiac pres-sures were normal. In the right and left ven-tricles of this small group of animals, the valuesfor total creatine, free creatine, CrP, Pi, andTASP closely resembled those found for normal,arrested hearts; the relationship between totalcreatine and CrP was also normal (Table IV,D, and Figure 2).

Normal skeletal muscles, when compared withnormal arrested hearts from the same animals,contained levels of total creatine, free creatine,and CrP that equaled those in the right ven-tricles and exceeded those in the left ventricles;levels of Pi and TASP in skeletal muscle werehigher than in either ventricle. The percentagesof total creatine present as CrP in normal skeletalmuscle equaled or exceeded the percentagesfound for the paired right ventricles. In skeletalmuscle samples removed from two animals withcongestive heart failure, the content of totalcreatine, CrP, Pi, and TASP remained withinthe normal range, although the hearts from theseanimals had shown significant decreases in totalcreatine and CrP (Table IV, E, and Figure 2).

Discussion

Previous studies by Davis and associates (30)indicated that in heart failure produced by pro-gressive pulmonary arterial stenosis, the rightventricle was predominantly involved, improve-ment followed digitalis therapy, and the syn-drome resembled severe heart failure in humanswith chronic pulmonic stenosis. In the presentstudy, strict criteria were required for the diag-nosis of heart failure. The elevated right ventric-ular end diastolic pressures might have reflecteddecreased compliance in the walls of the dis-tended and obstructed ventricles (39, 40), butthe association with breathlessness, circulatorycongestion, and elevations in systemic a-v 02difference indicated a relationship to myocardialfailure. Accurate assessment of left ventricularfunction in the failing hearts was difficult. Interminal right heart failure, a decrease in de-livery of blood to the left heart might have de-creased coronary perfusion and caused deterior-ation in function of both ventricles like that pro-duced by acute pulmonary arterial stenosis (41).The absence of dilatation and hypertrophy of theleft ventricles, and the rarity of elevations in leftventricular end diastolic pressures during markedsystemic circulatory congestion, suggested that de-spite the clinical appearance of breathlessness theleft ventricles were less compromised than theright.

Because of the great lability of ATP and CrPin the presence of the multiple degrading enzymespresent in samples of whole tissues, accurate esti-mates of true in vivo levels are difficult to obtain.Elaborate measures have been devised for in-stantaneous freezing of small biopsy samples ob-tained from skeletal muscles during a singletwitch (42) or from hearts beating in situ (43).The present study required relatively large sam-ples from both ventricles, and some hydrolysisof highly labile compounds, either CrP or un-identified intermediate products of oxidativephosphorylation, probably occurred during ex-tirpation. The values obtained for Pi served asrough indicators of phosphate bond hydrolysis.The levels of Pi found in the arrested hearts werehigher than those found in biopsy samples frozenin situ (26), but were equivalent to those foundin conventional biopsy samples or in perfused, ar-

213


rested hearts (27, 44, 45). Among the arrestednormal and hypertrophied hearts, the values forCrP in the left ventricles equaled those previ-ously reported (27, 44), and the values for theright ventricles were higher. The induction ofcardiac arrest allowed greater recovery of CrP,since contractions were not present during ex-tirpation and rigor during freezing was reduced.Levels of ATP in normal hearts removed whilebeating equaled those reported in other studiesusing careful techniques. Levels of ATP werenot increased by the induction of cardiac arrestbefore extirpation of the hearts, since ATP isless labile than CrP and can be transiently main-tained by the action of creatine transphosphory-lase. Considerable and constant artifactual hy-drolysis of ATP to ADP and AMP had notregularly occurred, since the proportions of ADPto total adenine nucleotides varied among differ-ent ventricles. A better indication that "free"ADP was not entirely derived from hydrolysisof ATP was provided by additional studies withradioactive phosphate, which demonstrated a dif-ference, under certain circumstances, between thedegree of labeling of the 8 phosphate of "free"ADPand the ,8 phosphate of ATP (46).

CrP levels in skeletal muscle were only slightlyhigher than in the arrested normal right ven-tricles. This finding conflicts with earlier reportsthat the content of CrP in skeletal muscle ex-ceeded that in the heart (7). The values foundfor Pi were higher than those found by precisemeasurements in cat and rabbit skeletal muscle(47), although they agreed well with valuesfound for rat skeletal muscle (48).

Since the values for CrP and for the percent-age of total Cr present as CrP in the normal rightventricles were generally higher than in the leftventricle, it was possible that the thicker leftventricles were more slowly arrested or frozen,allowing more time for hydrolysis of CrP. Ifsuch hydrolysis had occurred, higher levels of Piand of free creatine should have appeared in theleft ventricles. These levels were not increasedin the normal left ventricles, but were relativelygreater in left ventricles from hypertrophied andfailing hearts, suggesting some hydrolysis of CrP.

In the failing right ventricles removed in arrest,the decreased levels of CrP did not reflect increasedhydrolysis of CrP due to slower freezing of the

thick and dilated ventricles, since Pi and the per-centage of total creatine present as CrP were notchanged from the control values found for nor-mal or hypertrophied right ventricles.

Although the small decreases in concentrationsof ATP in the failing right ventricles were sig-nificant, the decreases in content of CrP wereclearly more impressive. The importance of CrPwas especially apparent in normal, arrestedhearts, where the high energy phosphate ac-counted for by the phosphagen was twice that inthe terminal phosphates of all nucleoside tri-phosphates. For use -in muscular contraction,the high energy phosphates stored in CrP mustbe transferred back to ATP, and it has been sug-gested that cytoplasmic creatine and CrP mayserve to shuttle phosphates between separate com-partments of ATP located at the mitochondriaand at the myofibrils (3). Since total creatineand CrP were decreased proportionately in thechronically failing hearts, it is possible that withlower levels of ATP, there may have been a sec-ondary decrease in synthesis of CrP or an in-crease in its utilization. The consequent in-crease in the fraction of free creatine that isreadily diffusible (49) might have produced agradual efflux of creatine from cardiac cells.In the present study, deliberate or accidental pro-duction of acute hypoxia during extirpation ofnormal or failing hearts caused hydrolysis of CrPbut no decreases in total creatine; -apparently anintact coronary circulation was required to re-move the increased amount of diffusible creatine.

Malnutrition, infection, hypoxia, cardiac fail-ure, and cardiac hypertrophy due to hypoxia havepreviously been reported to alter myocardial con-tent of creatine (50, 51). The first three of thesefactors were not overtly present in the heartfailure animals in this study, and it seemed likelythat an abnormality within the failing heartsthemselves, rather than changes in the systemicmetabolism of creatine, had decreased the myo-cardial levels of creatine and CrP. The decreasein creatine levels was not caused by congestionof the liver or kidneys, where creatine is princi-pally synthesized (52, 53), since concentrationsof total creatine and CrP were normal in thehearts of animals with hepatic and renal con-gestion produced by ligation of the thoracic in-ferior vena cava. The decrease in creatine in

214


the myocardium did not reflect a general depletionof creatine in all muscles, since skeletal musclesamples from two animals in congestive heartfailure contained normal amounts of creatine andCrP. A more specific control might have beenprovided by comparison of each heart with aconstantly active muscle such as the diaphragm,but technical limitations made this impossible.

It is unlikely that the decreases in content ofhigh energy phosphate compounds in the failinghearts reflected a simple physical dilution by anincreased proportion of protein in hypertrophiedmyocardial cells. Failing hearts of the type stud-ied here had previously been shown to containnormal amounts of actomyosin per unit of weight(54), and the hypertrophied control hearts in thepresent study showed no decreases from normallevels of creatine or CrP.

Since concentrations of UTP, GTP, and CTPdid not change significantly, within the wide limitsset by the control recovery studies, there wereprobably no marked alterations during congestiveheart failure in the metabolism of these compoundsor in their possible role in muscular contrac-tion (55).

The decreases found for ATP and CrP in thisstudy were similar to those observed by Feinsteinin left ventricles from guinea pig hearts failingwith aortic stenosis (28). The absence of suchchanges in other studies (11, 13, 16) may havebeen due to limitations in the methods employedfor tissue excision, extraction, or analysis or tothe fact that the ventricular failure studied wasnot so severe as that produced by isolated aorticor pulmonary stenosis. The present study indi-cates that within each terminally failing heart, themore severely stressed right ventricle showedgreater decreases in concentrations of ATP andCrP than did the left ventricle. Although bothventricles might have become chronically hypoxicas a result of decreases in cardiac output andcoronary perfusion, the dilated and obstructedright ventricles probably required greater oxygendelivery and utilization to support protracted in-creases in wall tension and time-tension index.

The decreases found for concentrations of ATPand CrP cannot definitively be related to the func-tion of the failing heart. The concentrations ofthese compounds were unchanged in the controlgroup of hypertrophied right ventricles although

heart-lung preparations exposed to acute systolicoverloading have shown marked decreases (56).The lower levels found for the failing hearts mightsimply have reflected a new steady state in whichthe energy sources available for muscular contrac-tion still remained adequate. In vitro modelshave demonstrated a coupling between mechanicalperformance of isolated skeletal myofibrils andmitochondrial generation of ATP and CrP (5).The deterioration in cardiac contractility asso-ciated with hypoxia or poisoning with digitalisglycosides has been only variably correlated withdecreased steady-state levels of high energy phos-phate compounds (14, 24), and such results can-not be properly extrapolated to the chronicallyfailing right ventricles in this study. The changesfound here were in hearts from animals in whichcongestive failure of maximal severity was takenas an end point. Producing failure in these prep-arations was difficult, and hearts for sequentialanalysis were not available from animals pro-gressing from mild to severe failure. It is there-fore not possible to speculate as to whether failurehad occurred after a gradual or an abrupt decreasein high energy phosphates or whether the chemi-cal changes were the cause, the result, or evenrelated at all to the severe mechanical failurepresent.

Nevertheless, the chemical changes found inthe chronically failing hearts can be usefully ex-amined in terms of the schema proposed bySiekevitz (6) in an attempt to elucidate thenature of the metabolic steady state present withinthe cells of the failing hearts. If it is assumedthat during severe failure, there is no change inthe basic amount of high energy phosphate re-quired to produce a unit of contractile force, theobserved falls in high energy phosphates mighthave reflected any of several possible abnormalmetabolic patterns: 1) normal ATPase activity atthe myofibrils associated with a primary defect inmitochondrial oxidative phosphorylation, 2) de-creased ATPase activity at the myofibrils asso-ciated with a greater decrease in mitochondrialoxidative phosphorylation, or 3) increased ATP-ase activity at the myofibrils and increased deliveryof ADP to mitochondria that were unable torespond with augmented ATP production. Themyokinase reaction, 2 ADP ATP + AMP,serves to maintain a constant ratio of ATP to

215


ADP when changes occur in the rate of produc-tion or utilization of either compound. Estimatesof the dominant direction of this reaction at equi-librium might be useful in determining whetherproduction of ADPwas indeed altered by markedchanges in myofibrillar ATPase activity. In fur-ther studies of normal and failing hearts (46), thestatus of oxidative phosphorylation was roughlyassessed by measuring the rate of incorporation ofradioactive phosphate into the terminal phosphateof ATP, and the activity of myokinase was evalu-ated by determining the rate at which the middlephosphate of ATP was labeled. These studiessuggested that the decreases found for concen-trations of ATP and CrP during severe, chronicfailure might be most compatible with an al-.tered metabolic state within the myocardial cellsin which a decrease in mitochondrial oxidativephosphorylation was associated with a decrease,as noted by others (19), in myofibrillar ATPaseactivity.

Summary

Congestive heart failure produced in dogs byprogressive stenosis of the main pulmonary ar-tery was more severe than the circulatory con-gestion produced by a combination of tricuspidinsufficiency and mild pulmonary arterial stenosis.Purine and pyrimidine nucleotides, creatine, cre-atine phosphate, inorganic phosphate, and totalacid-soluble phosphate were measured in the rightand left ventricles of normal, hypertrophied, andfailing hearts extirpated and frozen within 10seconds.

Among normal hearts, the right and left ven-tricles contained equal amounts of nucleotides,but the right ventricles contained 30% morecreatine phosphate. Induction of cardiac arrestbefore extirpation of the hearts improved recoveryof creatine phosphate. Hearts with right ven-tricular hypertrophy unassociated with failureshowed no changes in content of creatine or crea-tine phosphate.

With chronic heart failure due to pulmonaryarterial stenosis, right ventricles showed decreasesin adenosine triphosphate of - 12%b, in creatinephosphate of - 33 and - 43%o by two methods,and in total creatine of - 37%. The decreases inthese compounds were much less pronounced inthe paired left ventricles. Nucleoside triphosphates

other than adenosine triphosphate were not changedsignificantly. Hearts from animals with circulatorycongestion 'produced by ligation of the thoracicinferior vena cava, and skeletal muscle samplesfrom animals in heart failure, did not show de-creases from the normal levels of creatine or crea-tine phosphate.

The chemical changes found may or may nothave had a relationship to the mechanical per-formance of the failing heart, but might reflectan altered metabolic steady state within the myo-cardial cells.

Acknowledgments

The authors wish to thank Mrs. Marjorie Gable, Mrs.Madeline Britman, and Miss Jean Engel for their techni-cal help.

References1. Mommaerts, W. F. H. M., A. J. Brady, and B. C.

Abbott. Major problems in muscle physiology.Ann. Rev. Physiol. 1961, 23, 529.

2. Davies, R. E., D. Cain, and A. M. Delluva. Theenergy supply for muscular contraction. Ann.N. Y. Acad. Sci. 1959, 81, 468.

3. Carlson, F. D., and A. Siger. The mechano-chemistryof muscular contraction. J. gen. Physiol. 1960, 44,33.

4. Chance, B. The response of mitochondria to mus-cular contraction. Ann. N. Y. Acad. Sci. 1959, 81,477.

5. Watanabe, S., and L. Packer. Oxidative phos-phorylation of cardiac mitochondria in contrac-tion of glycerol-treated fibers of psoas muscle.J. biol. Chem. 1961, 236, 1201.

6. Siekevitz, P. Oxidative phosphorylation in musclemitochondria and its possible regulation. Ann.N. Y. Acad. Sci. 1959, 72, 500.

7. Olson, R. E. Physiology of cardiac muscle in Hand-book of Physiology. Washington, Amer. Physiol.Soc., 1962, vol. 1, p. 199.

8. Blain, J. M., H. Schafer, A. L. Siegel, and R. J.Bing. Studies on myocardial metabolism. VI.Myocardial metabolism in congestive heart failure.Amer. J. Med. 1956, 20, 820.

9. Goodale, W. T., R. E. Olson, and D. B. Hackel.Myocardial glucose, lactate and pyruvate metabo-lism of normal and failing hearts studied by coro-nary venous catheterization in man. Fed. Proc.1950, 9, 49.

10. Bing, R. J., E. Siegel, A. Vitale, F. Balboni, E.Sparks, M. Taeschler, M. Klapper, and S. Ed-wards. Metabolic studies on the human heart invivo; 1. Studies on carbohydrate metabolism of thehuman heart. Amer. J. Med. 1953, 15, 284.

216


11. Olson, R. E., and D. A. Piatnek. Conservation ofenergy in cardiac muscle. Ann. N. Y. Acad. Sci.1959, 72, 466.

12. Katz, L. N., H. Feinberg, and A. B. Schaffer. He-modynamic aspects of congestive heart failure.Circulation 1960, 21, 95.

13. Wollenberger, A. The energy metabolism of thefailing heart and the metabolic action of the cardiacglycosides. Pharmacol. Rev. 1949, 1, 311.

14. Furchgott, R. F., and T. de Gubareff. The highenergy phosphate content of cardiac muscle undervarious experimental conditions which alter con-tractile strength. J. Pharmacol. exp. Ther. 1958,124, 203.

15. Lee, K. S., D. H. Yu, and R. Burstein. The effectof ouabain on the oxygen consumption, the highenergy phosphates and the contractility of thecat papillary muscle. J. Pharmacol. exp. Ther.1960, 129, 115.

16. Buckley, N. M., and K. K. Tsuboi. Cardiac nu-cleotides and derivatives in acute and chronicventricular failure of the dog heart. Circulat. Res.1961, 9, 618.

17. Benson, E. S., B. E. Hallaway, and C. E. Turback.Contractile properties of glycerol-extracted musclebundles from the chronically failing canine heart.Circulat. Res. 1958, 6, 122.

18. Kako, K., and R. J. Bing. Contractility of actomyo-sin bands prepared from normal and failing humanhearts. J. clin. Invest. 1958, 37, 465.

19. Alpert, N. R., and M. S. Gordon. Myofibrillaradenosine triphosphatase activity in congestiveheart failure. Amer. J. Physiol. 1962, 202, 940.

20. Davis,.J. O., W. R. Carroll, M. Trapasso, and N. A.Yankopoulos. Chemical characterization of cardiacmyosin from normal dogs and from dogs withchronic congestive heart failure. J. clin. Invest.1960, 39, 1463.

21. Olson, R. E. The contractile proteins of heart muscle.Amer. J. Med. 1961, 30, 692.

22. Wollenberger, A., and W. Schulze. Mitochondrialalterations in the myocardium of dogs with aorticstenosis. J. biophys. biochem. Cytol. 1961, 10,285.

23. Gertler, M. M. Differences in efficiency of energytransfer in mitochondrial systems derived fromnormal and failing hearts. Proc. Soc. exp. Biol.(N. Y.) 1961, 106, 109.

24. Schwartz, A., and K. S. Lee. Study of heart mito-chondria and glycolytic metabolism in experi-mentally induced cardiac failure. Circulat. Res.1962, 10, 321.

25. Hurlbert, R. B., H. Schmitz, A. F. Brumm, and V. R.Potter. Nucleotide metabolism II. Chromato-graphic separation of acid-soluble nucleotides. J.biol. Chem. 1954, 209, 23.

26. Wollenberger, A., E.-G. Krause, and B. E. Wahler.Vber den tatsachlichen Orthophosphat- und N-Phos-

phorylkreatingehalt des Siugetierherzens. Pfluigers.Arch. ges. Physiol. 1960, 270, 413.

27. Fawaz, G., and E. Manoukian. Steady-state levelof phosphocreatine in the heart. Circulat. Res.1962, 11, 115.

28. Feinstein, M. B. Effects of experimental congestiveheart failure, ouabain and asphyxia on the high-energy phosphate content and creatine content ofthe guinea pig heart. Circulat. Res. 1962, 10, 333.

29. Barger, A. C., G. S. Richardson, and B. B. Roe.A method for producing chronic cardiac failurein dogs. Proc. Soc. exp. Biol. (N. Y.) 1950, 73,113.

30. Davis, J. O., R. E. Hyatt, and D. S. Howell. Right-sided congestive heart failure in dogs produced bycontrolled progressive constriction of the pul-monary artery. Circulat. Res. 1955, 3, 252.

31. Lee, Y. C. P., R. A. DeWall, and M. B. Visscher.State of creatine in mammalian heart muscle.Amer. J. Physiol. 1960, 198, 855.

32. Lilienthal, J. L., Jr., K. L. Zierler, B. P. Folk, R.Buka, and M. J. Riley. A reference base andsystem for analysis of muscle constituents. J. biol.Chem. 1950, 182, 501.

33. Oken, D. E., and R. J. Boucek. Quantitation of col-lagen in human myocardium. Circulat. Res. 1957,5, 357.

34. Deutsch, A., and R. Nilsson. Separation of adeno-sine and inosine phosphates by paper chromatog-raphy. Acta chem. scand. 1953, 7, 858.

35. Hawk, P. B., B. L. Oser, and W. H. Summerson.Practical Physiological Chemistry, 13th ed. NewYork, Blakiston, 1954, p. 953.

36. Furchgott, R. F., and T. de Gubareff. The determi-nation of inorganic phosphate and creatine phos-phate in tissue extracts. J. biol. Chem. 1956, 223,377.

37. Ennor, A. H., and H. Rosenberg. The determinationand distribution of phosphocreatine in animal tis-sues. Biochem. J. 1952, 51, 606.

38. Finlayson, J. K., M. N. Luria, and P. N. Yu. Somecirculatory effects of thoracotomy and intermit-tent positive pressure respiration in dogs. Circu-lat. Res. 1961, 9, 862.

39. Braunwald, E., S. J. Sarnoff, R. B. Case, W. N.Stainsby, and G. H. Welch, Jr. Hemodynamicdeterminants of coronary flow: effect of changesin aortic pressure and cardiac output on the rela-tionship between myocardial oxygen consumptionand coronary flow. Amer. J. Physiol. 1958, 192,157.

40. Braunwald, E., and J. Ross, Jr. The ventricular enddiastolic pressure. Appraisal of its value in therecognition of ventricular failure in man. Amer.J. Med. 1963, 34, 147.

41. Bacaner, M., J. E. Connolly, and D. Bruns. Thecoronary blood flow as a critical determinant ofcardiac performance and cardiac size. Amer. J.Med. 1961, 30, 392.

217


42. Mommaerts, W. F. H. M. Investigation of the pre-sumed breakdown of adenosine-triphosphate andphosphocreatine during a single muscle twitch.Amer. J. Physiol. 1955, 182, 585.

43. Wollenberger, A., 0. Ristau, and G. Schoffa. Eineeinfache Technik der extrem schnellen Abkuhlunggroserer Gewebestucke. Pflugers Arch. ges.Physiol. 1960, 270, 399.

44. Gott, V. L., M. Bartlett, D. M. Long, C. W. Lillehei,and J. A. Johnson. Myocardial energy substancesin the dog heart during potassium and hypothermicarrest. J. appl. Physiol. 1962, 17, 815.

45. Benson, E. S., G. T. Evans, B. E. Hallaway, C.Phibbs, and E. F. Freier. Myocardial creatinephosphate and nucleotides in anoxic cardiac arrestand recovery. Amer. J. Physiol. 1961, 201, 687.

46. Fox, A. C., and G. E. Reed. In preparation.47. Sacks, J., and R. S. Fulford. Free and bound or-

thophosphate and phosphocreatine in muscle. Arch.Biochem. 1964, 104, 423.

48. Threlfall, C. J. An analytical procedure for theacid-soluble phosphorous compounds in rat-skeletalmuscle. Biochem. J. 1957, 65, 694.

49. Lee, Y. C. P., and M. B. Visscher. On the state ofcreatine in heart muscle. Proc. nat. Acad. Sci.(Wash.) 1961, 47, 1510.

50. Myers, V. C. Some chemical changes in the myo-cardium accompanying heart failure. Bull. N. Y.Acad. Med. 1942, 18, 303.

51. Edman, C. D., N. P. Silvers, E. C. Gangloff, andR. F. Krause. Creatine, creatine phosphate, andcreatinine levels in hypoxia hypertrophied rathearts. Amer. J. Physiol. 1963, 204, 1005.

52. Ennor, A. H., and J. F. Morrison. Biochemistry ofthe phosphagens and related guanidines. Physiol.Rev. 1958, 38, 631.

53. Gerber, G. B., G. Gerber, T. R. Koszalka, and L. L.Miller. The rate of creatine synthesis in the iso-lated, perfused rat liver. J. biol. Chem. 1962, 237,2246.

54. Davis, J. O., M. Trapasso, and N. A. Yankopoulos.Studies of actomyosin from cardiac muscle ofdogs with experimental congestive heart failure.Circulat. Res. 1959, 7, 957.

55. Ranney, R. E. Biochemical characteristics of glycerolextracted myocardial fibers. Amer. J. Physiol.1955, 183, 197.

56. Hochrein, H., and H. J. D6ring. Die energiereichenPhosphate des Myokards bei Variation der Be-lastungsbedingungen. Pflugers Arch. ges. Physiol.1960, 271, 548.

SPECIAL NOTICE TO SUBSCRIBERS

Post Offices will no longer forward the Journal when you move.

Please notify The Journal of Clinical Investigation, BusinessOffice, 10 Stoughton Street, Boston, Mass. 02118, at once whenyou have a change of address, and do not omit the Zip Codenumber.

218

High Phosphate Compounds Myocardium Experimental ... · High energy phosphate compounds are...

Documents

Transcript of High Phosphate Compounds Myocardium Experimental ... · High energy phosphate compounds are...