Nucleotide Metabolism in Cardiac Activity
II. Reactions in Systole
By PHILIP A. KHAIRALLAH, M.D., AND W. F. H. M. MOMMAERTS, PH.D.
Relaxed mammalian hearts contain ATP as the predominant nucleotide, sometimes accompanied
by smaller amounts of ADP, presumably formed during manipulation. In systole, one-tenth of
the nucleotide is changed into a new compound, nucleotide H.
I
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N THIS STUDY (compare Khairallah and
Mommaerts5) we have investigated the nucleotide content of the resting heart* and
the breakdown of adenosine triphosphate
(ATP) in one single contraction. The background of this problem requires brief exposition.
After, in turn, lactic acid and phosphocreatine had been regarded as the contraction-causing substance in voluntary muscle, f this role
was finally assigned to ATP by Lohmann6 because of the observation that muscle contains
no enzymes capable of dephosphorylating phosphocreatine directly. Instead there are enzymes
which, in the so-called Lohmann reaction,
transfer a high-energy phosphate radical from
phosphocreatine to ADP (or AMP), whereupon, through breakdown of the ATP formed,
a net dephosphorylation of ATP results, f In
this way, ATP became, by exclusion, regarded
as the ultimate substrate for the shortening
reactions. While this view received spectacular
support by the discovery of ATP-actomyosin
interactions, f the actual dephosphorylation of
ATP during contraction still remains to be
demonstrated (Hill); furthermore, it was found
that the physical effect of ATP upon actomyo-
sin systems may not be dependent on the decomposition of the former.7
The present publication considers this problem for the case of cardiac muscle. This work
is based upon preparatory studies on the
nucleotide content of this tissue reported in
the preceding paper,6 and aims at the determination of the ATP breakdown occurring in one
single contraction.
METHODS
Analytic Technic. The methods of extraction and
tissue analysis were described in the previous paper.5
Relaxed Hearts. Female cats were anesthetized
with nembutal or, preferably, with ether. The trachea was intubated, the chest opened, and positive
pressure respiration was applied. It was attempted
to effect diastolic arrest by electric stimulation of
the peripheral ends of the severed vagi; but, due to
escape before the heart could be excised, this method
was usually not successful. The procedure finally
adopted was to inject into the femoral vein 10 ml.
of isotonic potassium chloride containing 6 mg.
physostigmine and 100 mg. acetyl-/3-methyl choline.
This produced a prolonged diastolic arrest, sufficient
for excision of the heart and transfer into a large
volume of isotonic potassium chloride at room temperature (about 30 C). The heart would remain
inactive or perform up to three contractions before
arrest in diastole. It was then rapidly extracted and
worked up as described.
Contracted Hearts. Relaxed feline hearts, obtained
as described in the previous paragraph, were dropped
into an acetone-dry ice mixture or into liquid air,
which caused sudden contraction and immediate
freezing in the contracted state. In other experiments, dogst under ether anesthesia were rapidly
cardiectomized without further preparation, and
the hearts immediately dropped into the cold mixture as described. The dog hearts were stored at
From the Department of Biochemistry, Duke
University School of Medicine, Durham, N. C.
This investigation was supported by Research
Grant No. H229 from the National Heart Institute
of the National Institutes of Health, U. S. Public
Health Service.
This work was done during the tenure of a Postdoctoral Research Fellowship, and an Established Investigatorship of the American Heart Association,
respectively.
Received for publication Oct. 1, 1952.
* See the discussion for a validation of the use
of this term.
f See Mommaerts8 for a historical introduction.
t We are indebted to Dr. J. W. Beard of the Department of Surgery for his cooperation in making
available to us a number of the animals.
12
Circulation Research, Volume /, January 1959
13
PHILIP A. KHAIRALLAH AND W. F. H. M. MOMMAERTS
—20 C. prior to analysis, the cat hearts could be
analyzed immediately. The hearts were crushed in
the frozen state and extracted.
This procedure to obtain contracted hearts is
based upon the observation by Embden2 that a
skeletal muscle thus treated is fixed in the contracted condition. Admittedly, this procedure is
unphysiologic; however, other methods of stimulation are no less artificial, whereas the present method
0.003 N HCL
0 01 N HCL +
0 0 2 M NoCl
1
1
0 01 N HCL +
0 2 M NQCI
05-
ments, such amounts of ADP occurred five
times; these hearts were, to all appearances,
perfectly relaxed, but after isolation had performed two or three contractions before arrest.
In those hearts (three out of eight) which conTABLB 1.—Nucleotide Distribution of Relaxed Hearts,
Expressed as Percentage of Total Nucleotide
AMP
IMP
NH
ADP
ATP
0
0 0
0 0
0 0
0
0
0 0
0 0
0 0
0
0
0 0
0 0
0 0
0
29 .0
0 21. 0
0 29. 2
0 38. 6 32 .7
71 .0 100 79. 0 100 70. 8 100 61. 4 67 .3
0 4-
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0.003 N HCL
0.3-
001 N HCL +
0 0 2 M NoCl
' v 001 N HCL +
1
0 2 M NoCl
o
0.2-
0.1 -
n •
TUBE NUMBER
k
FIG. 1
0 01 N HCL +
0 02 M NoCl
z
° 1.0
§
001 N HCL •+
02 M NoCl
TUBE NUMBER
FIG. 3
£ 04
001 N HCL +
Q02 M NoCl
001 N HCL +
0 2 M NoCl
n~m-n^_
TUBE NUMBER
2
FIGS. 1 AND 2. Nuoleotide chromatograms of "reFIG.
g 06-
3
laxed" hearts. In figure 1, no ADP is present, in
figure 2, ADP occurs.
has the advantage of immediately interrupting all
chemical reactions. Other approaches to this problem are being investigated.
RESULTS
Relaxed Hearts. Two examples of chromatographic analyses are given in figures 1 and 2.
In the first case, ATP was the only adenine
nucleotide present. In the other example, some
ADP was present, and in such cases this usually
amounted to about 20 or 30 per cent of the
total nucleotide (table 1). Out of eight experi-
_m
ifl LL.
TUBE NUMBER
FIG. 4
FIGS. 3 AND 4. Nucleotide chromatograms of con-
tracted hearts. In figure 3, no ADP is present, in
figure 4, ADP occurs. Note the presence of a component eluted by solvent A.
tained no ADP, no visible contractions had
been observed between excision and extraction.
Contracted Hearts. Examples of analyses of
hearts extracted after freezing in systole are
given in figures 3 and 4. As in the case of
14
NUGLEOTIDE METABOLISM IN CARDIAC ACTIVITY
relaxed hearts, either no or about 20 per cent
ADP was found in addition to ATP (table 2).
In addition, however, all contracted hearts
showed the presence of a new component, eluted
by solvent A5 later than AMP and IMP would
be expected. According to its absorption spec
trum it is an adenine compound, and it was
always found to constitute one-tenth of the
total nucleotide (table 2). This substance,
which will be called nucleotide H, was found to
contain two atoms of phosphorus and 1 mole
of ribose per mole of adenine, and an excess of
nitrogen over that present in the adenine
moiety.
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TABLE 2.—Nucleotide Distribution of Contracted
Hearts, Expressed as Percentage of Total Nucleolide
AMP
IMP
NH
ADP
ATP
0
0
9 .8
0
90 .2
0
0
10 .3
24 .4
65 .3
0
0
11 .1
20 .7
6S .2
0
0
10 .4
18 .6
71 .0
0
0
9. 0
18. 1
72. 2
0
0
9. 7
21. 8
6S. 5
DISCUSSION
Although the data presented in this paper
need considerable elaboration both in physiologic and in chemical respects, we believe that
certain conclusions emerge quite clearly.
With respect to the "relaxed" or "resting"
heart, we must admit the arbitrariness of such
an expression. While the concept of a resting
skeletal muscle is a justifiable extrapolation,
the myocardium never rests. We believe, however, that a heart in diastole must, in structural
and biochemical respect, be quite comparable
to an unstimulated resting muscle. The analogy
between a contracted muscle and a heart in
systole, or artificially caused to contract, is then
obvious.
In the relaxed myocardium thus defined we
have detected sometimes exclusively ATP,
sometimes ATP with some ADP. Although
more observations are necessary to definitely
establish this correlation, it is suggested by our
observations that all the nucleotide is in the
form of ATP if the heart remained inactive
throughout the manipulation; whereas, ADP
is found in those cases where a few contractions
took place before the heart stopped in diastole.
In the contracted hearts we find the same
variability with respect to the occurrence of
ADP besides ATP, but in addition we note the
appearance of a new compound, nucleotide H,
constituting 10 per cent of the total nucleotide.
It is proposed, therefore, that the relaxed heart
fundamentally contains only ATP, that the
presence of ADP is a consequence of a few
twitches under abnormal conditions (see below), and that the conversion of one-tenth of
the ATP into nucleotide H is a characteristic of
the contractile process itself. We have, so far,
obtained no evidence of the appearance of
nucleotide H in the activity of skeletal muscle.
That the performance of a few contractions
during the isolation leads to the accumulation
of ADP (corresponding roughly to a conversion
of one-tenth of the ATP for each twitch), when
in normal activity any decomposed ATP
must be resynthesized, may perhaps be ascribed to the adverse conditions during manipulation. This is quite acceptable for those cases
where the isolated heart is kept in isotonic
potassium chloride, the relaxing influence of
which may be related to the indirect or direct
inhibition of certain enzymes. The explanation
is less obvious for the dog hearts which were
frozen immediately after extirpation. Here, due
to the large size of the hearts, it is possible that
incompletely reversed activity occurs in central
parts of the organ during freezing. It remains
possible, however, that the presumed correlation with the number of postisolative contractions is fallacious, and that an isolated heart
contains between 0 and 30 per cent of the nucleotide in the form of ADP, dependent on the
carefulness of the manipulations.
If, indeed, systole is correlated with a breakdown of 10 per cent of the ATP into nucleotide
H, it follows from the ATP content that the
complete contraction of 1 Gm. of cardiac muscle
involves the metabolism of 5 X 10~7 moles of
ATP. From the respiratory rate of a maximally
contracting heart, it may be calculated that
this amount of ATP is dephosphorylated in
each cardiac cycle.* The same number has also
* Under conditions of average activity and frequency, the mammalian heart consumes some 300 to
400 ml. oxygen per hour per 100 Gm. tissue. 3 If this
respiratory metabolism serves to resynthesize any
PHILIP A. KHAIRALLAH AND W. F. H. M. MOMMAERTS
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been computed8 and approximately determined12 for skeletal muscle, where it is quantitatively accounted for by the polymerization of
actin.0-11
The present results do not yet warrant any
proposals regarding the chemical reactions occurring during contraction. We have preliminary evidence that nucleotide H is a compound
of adenosine with two phosphate radicals and a
glutamic acid or glutamine peptide. This would
have most interesting implications in view of
the striking but unexplained occurrence of glutamine in the heart,1 and because of our contention8 that the breakdown of ATP may not
be a direct hydrolysis but may require a coenzyme or activating cosubstrate. The relation of
nucleotide H to molecular processes involving
actin or actomyosin will be investigated.
SUMMARY
1. The adenine nucleotide contents of relaxed and contracted myocardia have been
compared.
2. The relaxed heart contains mainly or exclusively ATP; sometimes some ADP is found.
3. In the contracted heart, one-tenth of the
nucleotide is found to be converted into a new
component, which contains adenine, ribose,
phosphate and additional nitrogenous constituents.
ATP split us a consequence of contractile activity,
this number indicates that ATP is utilized to the
extent of 2 to 2.5 X 10~7 moles per gram per cycle
(see reference 8, chapter III, for such calculations).
In maximal activity, respiration is about doubled,3
so that complete activation of the contractile structure (as apparently occurs in our experimental procedure) involves 5 X 10~7 moles ATP per gram.
15
4. It is calculated from physiologic data that
in intense activity up to one-tenth of the ATP
is dephosphorylated and resynthesized in each
cardiac cycle.
REFERENCES
1
ARCHIBALD, R. M.: Chemical characteristics and
physiological roles of glutamine. Chem. Rev.
37: 161, 1945.
2
EMBDEN, G., AND LAWACZECK, H.: Tiber die Bildung anorganischer Phosphorsaure bei den Kontraktion des Frosch muskels. Biochem. Ztschr.
127: 181, 1922.
3
EVANS, C. L.: The metabolism of cardiac muscle.
In Newton, W. H.: Recent Advances in Physiology, Philadelphia, Blakiston, 1939. Vol. 6, p. 157.
4
HILL,, A. V.: A challenge to biochemists. Biochim.
et biophys. acta 4: 4, 1950.
6
KHAIRALLAH, P. A., AND MOMMAERTS, W. F.
H. M.: Nucleotide metabolism in cardiac activity: I. Methods and Initial Observations.
Circ. Res. 1: 8, 1953.
c
LOHMANN, K.: Uber die enzymatische Aufspaltung
der Kreatin phosphorsaure; Zugleich ein Beitrag
zum Chemismus der Muskelkontraktion. Biochem. Ztschr. 271: 264, 1934.
7
MOMMAERTS, W. F. H. M.: The reaction between
adenosine triphosphate and myosin. J. Gen.
Physiol. 31: 361, 1948.
8
—: Muscular Contraction, A Topic in Molecular
Physiology. New York, Interscience Publishers,
1950. Pp. 39-40, 94-95.
9
—: A consideration of some experimental facts
pertaining to the primary reaction in muscular
activity. Biochim. et biophys. acta 7: 477, 1952.
10
—: Phosphate metabolism in the activity of skeletal and cardiac muscle. In McElroy, W. D., and
Glass, B., eds.: Phosphorus Metabolism. Baltimore, Johns Hopkins Press, 1951.
11
—: The molecular transformation of actin. III.
The participation of nucleotides. J. Biol. Chem.
198: 469, 1952.
12
—, AND RUPP, J. C : Dephosphorylation of ade-
nosinetriphosphate. Nature 168: 957, 1951.
Nucleotide Metabolism in Cardiac Activity: II. Reactions in Systole
PHILIP A. KHAIRALLAH and W. F. H. M. MOMMAERTS
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Circ Res. 1953;1:12-15
doi: 10.1161/01.RES.1.1.12
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