Effect of Temperature on the Heart Rate,Electrocardiogram and Certain Myocardial Oxidations

of the RatBy JOHN P. HAXXOX, P H . D .

Deep hypothermia in the rat was studied with respect to the sequential changes in bodytemperature, heart rate, and electrocardiogram. In vitro assays of myocardial metabolicactivity were made at temperatures ranging from 5 to 35 C. These assays stronglysuggested that the in vivo cardiac dysfunctions observed at low temperatures wereattributable to shifts in temperature kinetics of enzyme systems at 20 to 21 C.

IX A recent investigation1 of the in vitrometabolism of ventricular tissue from

hypothermic rats, no irreversibly damagingeffects were observed. Instead, lowering thebody temperature to 15 C. led to an increasedendogenous metabolism when subsequentlymeasured at 38 C. It was concluded that theonly metabolic effect of hypothermia on therat heart was a possible mild hypoxia and anincreased permeability to substrates. It wasconceived that the hypoxia led to an accumu-lation of endogenous substrate and subse-quently to an increased endogenous respira-tory rate.

During a review of the literature for thestudy just mentioned, 2 observations weremade. The first was that little use has beenmade of the laboratory rat for deep hypother-mic heart studies, and the second was thatlittle effort has been made to study the metab-olism of the mammalian heart tissue while itis under the influence of hypothermia.

As a result of these observations the experi-ments to be described here were undertakenwith 2 purposes in mind. The first was tooutline generally the response of the rat tostandardized hypothermia. In this regard,heart rates, cooling times, and eleetrocardio-graphic measurements were made. The s.v-ond purpose of these experiments was to assaythe response of certain aerobic-enzyme sys-tems to various temperatures and to deter-

Froin the Biochemistry Branch, Arctic Aeromcrli-r:il Laboratory, APO 731, Seattle, Wasli.

Kceeivpfl for publication Mny 23, 195S.

mine whether there were any relationship be-tween in vitro metabolic activity and in vivomyocardial response to hypothermia.

METHODS

Male rats of the Sprague-Dawley strain weigh-ing between 300 and 375 grams were used in allexperiments. They were maintained on a diet of'•Friskies" dog food and water, fed ad libitum,for at least a month prior to experimentation.

In experiments where hypothermia of the intactanimals was studied the rats were first anesthetizedby intraperitoneal injection of sodium thiopental(40 to 60 mg./Kg. body weight). Following ad-ministration of the anesthesia, the rats were tiedin a prone position with legs extended to a smallboard placed at an angle of about 30 degrees fromthe horizontal. This board was then placed in anice and water bath of 1 to 3 C. The level ofimmersion was adjusted to extend from just underthe forelegs and lower chest to over the lumbarregion of the back. Temperature was recordedby a "thermistor" rectal probe carefully insertedto a depth approximating that of the caudal partsof the liver. Care also was taken to place thisprobe as near as possible to the core of the animal,as preliminary experiments utilizing intraventricu-lar thermocouples had shown that this positiongave a close approximation of heart temperaturesin deep hypothermia.

Electrocardiograms were recorded on a SanbornVisocardiette, using the number I lead positionand a tape speed of 25 mm./sec. The leads wereadapted to take no. 20 syringe needles. Thesewere inserted ju.st under the skin on either sideof the chest. In addition, when a continuousrecord of heart rate throughout the whole hypo-thermic episode was desired, electrocardiogramswere made on a Sanbnrn model no. 150 recorderset at tape speeds ranging from 10 nun.'sec. to 1mm./sec.

Circulation Rrtrnrrh. Volttmr VI, Novrmbrr

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772 HANNON

so » toBODY TEMPERATURE (*C)

Fio. 1. Body-cooling patterns of rats during hypo-thermia. A. Time in minutes to reach various hypo-thermic body temperatures. B. Differential coolingrate as time in minutes/degree decline in body tem-perature at various levels of hypothermia.

In experiments where ventricular metabolic rateswere determined, normothermic animals were firstkilled by a blow on the head and the heart quicklyexcised and chilled in chopped ice. The atria andA-V septurns were trimmed away from the ven-tricles and the excess blood was pressed out.Following this, the ventricles were weighed on amicrotorsion balance and placed in a Potter-Blvehjem type of homogenizer.2 An ice-cold, 10per cent homogenate was prepared in a mediumconsisting of 0.25 M sucrose, 0.0001 M ethylenediamine tetra-acetie acid (pH 7.4) and 0.01 MTi-is (hydroxymethyl) amino methane buffer (pH7.4). After filtering through 4 layers of cheesecloth to remove any large tissue fragments thehomogenates were diluted to 3 1/3 per cent withcold homogenizing medium. One-milliliter aliquotswere taken from the final dilution and placed inthe reaction vessels for the metabolic rate measure-ments.

The incubation medium for measuring metabolicrates had the following constituents: 0.002 M K-ade-nosine-triphosphate (pH 7.4), 0.006 M MgCl2, 1.5X10"5 M cytochrome c, 5 X KH M diphophopyri-dine nucleotide, 0.01 M K-phosphate buffer and0.01 M snccinate, or a mixture of 0.01 M pyruvatennd 0.01 M malate. Substrates, where used, werein the form of the potassium salts. The finalvolume of the medium and homogenate in eachreaction vessel was adjusted to 3.0 ml. with 0.25M sucrose. Isotonie conditions were thus approxi-mated.

Following a five-minute thermoequilibration per-iod, incubations were conducted for 30 min.,according to standard manometrie procedures.2

The gas phase in each reaction flask was air.Carbon dioxide was absorbed by 0.2 ml. of 5NXaOH placed in the center well along with a foldedpiece of filter paper. In a series of separate ex-

periments, oxygen eonsuinption was measured attemperatures of 5, 10, 15, 20, 25, 30 and 35 C.The arithmetical average of 3 animals was usedto establish each point.

Metabolic rate was calculated over five-minuteintervals during the course of incubation and isexpressed as /u.1 oxygen consumed per mg. oftissue. In addition, the respiratory rates over 30min. of incubation were used for Arrhenius-van'tHoff plots and the calculation of temperature-velocity constants.

These constants were calculated according tothe following formula :

hi h. = JL ( J_ _ J_ >fcs R \ T2 T, )

where jx represents the temperature-velocity con-stant, fe, and fc2 the reaction rates at absolutetemperatures T, and T2 respectively, and B thegas constant in calories.

RESULTS

Hypothermia in the Intact Rat

The first series of experiments was designedto characterize some of the effects of hypo-thermia in the rat. Of particular interestwere the body cooling rates under a stand-ardized immersion, the changes in heart rateduring the course of hypothermia and some ofthe electrocardiographic anomalies exhibitedby the animals.

Figure 1 gives a summary of the data onaverage body-cooling rates gained from 9animals under the conditions described (see"Methods"). In this figure, curve A showsthe average time to cool from a body tempera-ture of 35 C. (cooling occurred a few minutesafter immersion in the 1 to 3 C. bath) to vari-ous body temperatures as low as to 15 C.Standard deviations of the mean are shownat 5 degree intervals during the course of cool-ing. Curve B in figure 1 indicates the differ-ential cooling rate of these rats in rnin./degreefall in body temperature from 35 to 15. Hereit can be observed that very early in the cool-ing process, i.e., while the body temperatureis between 35 and 34, there is a relativelyslow rate of cooling. This is followed by asharp increase as the body temperaturereaches about 30 C. Thereafter the differen-tial cooling rate becomes progressively sloweras body temperatures approach 15 C. Curves

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TEMPERATURE AND MYOCJARDIAL OXIDATIONS 773

35 30 25BOOY TEMPERATURE CO

20

FIG. 2. Chaiiges in heart rate of lightly anesthetizedrats, A, and deeply anesthetized rats, B, during hypo-thermia.

A and B taken together, therefore, character-ize the nature of the cooling process in theadult Sprague-Dawley rat.

The second characteristic studied in thehypothermia rat was heart rate. The resultsof this study are presented in figures 2 and 3.In figure 2, curve A gives the changes in heartrate in the lightly anesthetized, violentljr shiv-ering animal. These data were taken from 3individuals with the range of the 3 values atvarious body temperatures indicated by ver-tical lines. Curve B gives the average heartrates of 8 deeply anesthetized animals duringthe course of hypothermia. Here the verticallines represent the standard deviations of themeans. The obviously more scattered data inthe latter curve are felt to be a reflection ofthe absolute depth of anesthesia in these ani-mals. In agreement with this was the obser-vation of occasional mild shivering in some ofthe animals used in obtained data for curve B.

In both curves of figure 2, it is importantto note the sharp decrease in heart rate atbody temperature of 20 to 21 C. The implica-tions of this change will be noted later in re-lation to the alterations in myocardial meta-bolic rates at low temperatures.

Following the break at 20 to 21 C. the heartrate, aside from being much lower, became

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Fia. 3. Two typical examples of the fluctuations inheart rate of rats during deep hypothermia.

quite erratic, showing considerable variabilityfrom animal to animal. Since there were un-predictable degrees of A-V blockade and ar-rhythmia, rates beyond this point were calcu-lated from only the ventricular portions ofthe electrocardiograms. Some impression ofthe variability in the ventricular rate fromthe breaking point onward can be gained fromfigure 3. Here, the rates of 2 typical animalsare plotted at 0.2 C. intervals from body tem-peratures of 22 down to 13. These plots, al-though giving a fairly accurate indication ofthe break in the rate curve, still do not givea completely true picture of the abnormalitiesat lower temperatures. This is because theplotting interval of 0.2 degree used here istoo coarse to reveal the finer details of therate fluctuations. In addition, at times it isvirtually impossible to tell from an electro-cardiographic tracing whether all of the fluc-tuations actually represent contractions.

Some indications of the difficulty encoun-tered in making rate measurements at lowertemperatures can be seen in figure 4, showingelectrocardiographs changes typical of thosefound in the rat. They were recorded at thevarious body temperatures indicated. Besidesthe variability of the ventricular rate at tem-peratures below 20 to 21 C., shown by thesetracings, other abnormalities include: shiver-ing patterns at temperatures down to 21, A-ar-iable degrees of A-V blockage, abnormal andquite often bizarre ventricular complexes, andoccasional ventricular tachycardia. Ventricu-lar fibrillation was not observed.

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774 HAXXOX

Fia. 4. Typical examples of tlic electrotiirdiogrupliie changes in Hie rat during liypotliermin.

Myocavdial MetabolismThe second serie.s of experiments in the

present study was concerned with the in vitroactivity of certain myocardial enzyme sys-tems at different temperatures. The resultsof the first of these experiments is shown infigure 5, where suecinate oxidation was meas-ured at 5 degree intervals between 5 and 35C. In the left-hand portion of this figure theseries of curves shows the oxygen consump-tion of the tissue at the various incubationtemperatures. As would be expected, the rateof succiuate oxidation increases with temper-ature. In the right-hand portion of the fig-ure an Arrhenius-van't Hoff plot of the datahas been made. That is, the logarithm of therate of oxidation has been plotted against thereciprocal of the absolute temperature. Inviewing this plot it should be rememberedthat temperature decreases from left to right,each point being 5 degrees from its neighbor,

In these data, a sharp break in the rate ofoxidation occurs at a temperature of 20 to 21C. Above this, the temperature-velocity con-stant indicated by A has a value of 8,490 cal-ories whereas below this point, indicated by B,the constant has more than doubled to a valueof 19,960 calories. In other words, what issometimes called the "activation energy" ofthe system required for suecinate oxidationshows a sharp increase at temperatures lowerthan 20 to 21 C.

Another, and perhaps more familiar, wayof viewing these data is from the standpointof the Qio values. AVhen these are calculated,portion A of the Arrhenius plot has a valueof 1.59 and portion B has a value of 3.33. Thusthe change in rate of suecinate oxidation per10 degree change in temperature is at leasttwice as great below tiie 20 to 21 transitionpoint as above it.

Similar experiments utilizing other sub-

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TEMPERATURE AND MY0CABD1AL OXIDATIONS 775

strates also revealed sharp breaks in the tem-perature-velocity constant curves in theneighborhood of 20 C. However, in thesecases the Arrhenius-van't Hoff plots werecurvilinear below the critical point, with atrend toward gradually increasing tempera-ture-velocity constants as the incubation tem-perature became lower. This will be observedin figure 6, where the rate of endogenous res-piration is depicted. For convenience, since,the curve below the break does not yield asingle value, the lower portion was dividedinto 2 sections: one, from 20 to 10 is signifiedby the B -. the other, from 10 to 5 by the letterC. From this, temperature-velocity constantswere calculated to be 13,520 calories for A,27,580 calories for B, and 34,920 calories forC. Expressed as Q,,,, .4. would be 2.08; B.5.24; and C, 9.17.

Figure 7 shows data gained from anotherof these experiments. Here the respiratoryrate is seen measured as it might occur underconditions of carbohydrate metabolism, thatis, in the presence of pyruvate, an end prod-uct of glycolysis, and malate, a component ofthe tricarboxylic acid cycle. Under normalconditions pyruvate is converted to acetyl co-enzyme A and this in turn is condensed withoxalacetate to form citrate. In the presentexperiment, malate was chosen in preferenceto oxalacetate because of the chemical insta-bility of the latter. Tn living systems themore stable malate is rapidly converted tooxalacetate thus assuring an adequate sub-strate level for the condensation reaction withacetyl coenzyme A.

In the Arrhenius-van't Hoff plot of theright of figure 7 it can be seen that the tem-perature response of the tissue in the pres-ence of these 2 substrates resembles that ob-served previously, when only endogenous res-piration was measured. In fact, the temper-ature-velocity constants were quite similar:10170 calories for .4, 26,300 for B, and36,570 for C. This corresponds to Q10 valuesof 1.74, 5.02 and 10.02 respectively. Andagain, there is a marked shift in the tempera-ture-velocity constant at the breaking pointnear 20 C.

FIG. 5 Top. Effect of temperature on suceinnte oxi-dation by rut heart houiogenutes. Left. Pattern ofoxygen uptake at various incubation temperatures.Right. Arrliciiiiis-vnn't Hoff plot of oxygen uptake.

Fio. 6 Bottom. Effect of temperature on ondo-gcnoiix motaboligm of rat heart homogenates. Left.Pattern of oxygen uptake at various incubation tem-peratures. Riglii. Arrhenius-van't Hoff plot of oxy-gen uptake.

DISCUSSION

The experiments reported here have shownthat the response of the rat to hypothermia isquite similar in many respects to that ob-served in larger mammals, and in many re-spects similar to the responses in rats pre-viously observed by Crismon.:t Accordingly,the fall in body temperature during a stand-ardized immersion procedure, although some-what more accelerated than that observed inanimals such as the dog, follows the samegeneral pattern; i.e., a relatively slow initialdecline which appears to correspoud with the''summit metabolism" discussed by Kayser.'1

This is immediately followed by a more rapidfall until body temperature reaches 30 to 31C. whereupon the rate of decline graduallydecelerates as hypothermia progresses. A sec-ond similarity between the larger mammals

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776 HANNON

D O ID »IWO*ATKJN TlWt (HIHj

FIG. 7. Effect of temperature on tlie oxidation ofpyruvate and malate by rat heart homogenates. Left.Pattern of oxygen uptake at various incubation tem-peratures. Arrhenius-vHii't Hoff plot of oxygen up-rnko.

and the hypothermia rat is the behavior of theheart rate. The rate, although not followingexactly the van't Hoff-Arrhenius rule as re-ported by Kayser5 and Meda,9 neverthelesswas shown to exhibit a sharp break in theneighborhood of 20 to 21 C. This appears tobe in approximate agreement, from the stand-point of temperatures, with the onset of se-vere bradycardia and fibrillation observed inthe dog,7-8 but a little lower than reportedfor the guinea pig.6

Finally, the electrocardiogram of the rat indeep hypothermia exhibits many characteris-tics similar to those of other animals. Amongthese are partial and complete A-V blocks,arrhythmias and bizarre patterns. The onlyreal difference is the absence of ventricularfibrillation in the rat. This difference appearsto be secondary to the basic biochemicalchanges within the tissue that lead to theabrupt fall in rate at around 20 to 21 C. Theabnormalities in cardiac function after thispoint, whether they be arrhythmia, A-Vblocks or fibrillation, probably can all betraced ultimately to the same initial sourceas that leading to the rate discontinuity.

Most, if not all, of the cardiac functionsadversely affected by deep hypothermia aredependent upon endergonic chemical activity.That is, they require an input of energy. Itseems only reasonable, therefore, that one ofthe first places to look for the cause of cardiacdysfunction at low temperature would be at

the source of this energy, viz., at the exer-gonic reactions within the tissue where energyis released. In this regard, the reactions ofthe tricarboxylic acid cycle are of primaryimportance since they are the major sites ofmetabolic energy release.

The metabolic studies reported here are theresult of this reasoning, and, although some-what general in scope, they appear to havegiven a key to the cause of the functionalabnormalities observed at low temperature.This key is the similarity in temperature levelfor the onset of in vivo cardiac malfunctionand the in vitro break in myoeardial oxidativemetabolism when the latter is plotted accord-ing to the Arrhenius-van't Hoff formula.

In view of the simultaneous breaks at about20 to 21 C. in the Arrhenius van't Hoff plotof several different tricarboxylic acid cycleoxidations, it is legitimate to ask what suchbreaks mean from a physiological standpoint.It is obvious from the data given here that thedecline in oxidative rate with a given decreasein temperature is at least twice as great below20 to 21 C. as above it. Therefore it can beconcluded that there is a much sharper reduc-tion per the degree the temperature is low-ered, in the amount of energy made availablefor physiological processes of the tissue attemperatures below this point than above it.

When viewed from the standpoint of thetemperature-velocity constant, /t, the doublingof the value below the breaking point, is some-what difficult to interpret. If the p. valuewere representative of a simple chemical reac-tion it would correspond to the activationenergy of the reaction. In such cases, a doub-ling of the constant would indicate a doublingof the kinetic energy of the reacting mole-cules before the reaction could proceed. Morespecifically, in the case of a simple, enzyme-catalyzed reaction, the /* value is approxi-mately equal to the change in heat content,AH, when the activated complex is formedfrom reactaut molecules.9

Biological oxidations such as those reportedhere are not as simple as this, however, sincea whole chain of enzyme-catalyzed reactionsis usually involved. In these multienzyme

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TEMPERATURE AND MYOCARDIAL OXIDATIONS 777

systems the fx value may represent the processas a whole. Accordingly, it could result fromdominance shared by a number of reactions,each with different temperature characteris-tics, as suggested by Burton.10 On the otherhand, one step within the system may be rate-limiting and the constant could reflect thekinetics of this reaction alone.

Carrying this latter reasoning a step fur-ther, Crozier11 has postulated that the slowestreaction in a sequential series of reactions isthe pacemaker or master reaction and hencedetermines the /x value for the over-all rate.A shift in the ^ value is then interpreted asindicating that a different step in the sequenceof reactions has become the pacemaker. It isnoteworthy, with respect to shifts observed inthe present studies, that Hadidian and Hoag-land,12 working with beef heart succinoxidasepreparations found a JX value of 11,200 cal-ories for the suceinic dehydrogenase portionof the oxidation and a value of 16,000 caloriesfor the cytoehrome-cytochrome oxidase por-tion. In addition, they were able to shift theix value of the system as a whole from 11,200calories to 16,000 calories through the addi-tion of cyanide to inhibit the oxidase portion.A similar shift, although obviously not causedby cyanide, could be responsible for thechange in /x values of myocardial metabolism.

However, in the present studies it must beremembered that a number of enzymes areactive in each of the experiments and the ques-tion of which of these, if any, are the pace set-ters at the different levels of hypothermia willhave to await additional studies. The proba-bility of one master reaction establishing therate above, and another the rate below, thecritical temperature of 20 to 21 C. could beconsidered a debatable point on the basis ofthe magnitude of shifts in /x value observedhere.10

Finally, it is possible that the same enzymeis controlling the rate of the oxidations at alltemperature levels. If this were true, thechange in fx value could be a reflection ofsome physical or chemical alteration of theprotein configuration of this particular en-zyme, as has been suggested by Belehradek,1"

and Kavanau,14 or by a reversible heat inac-tivation above the critical temperature, assuggested by Morales.15

SUMMARY

With rats as experimental animals, studieswere conducted to establish the nature of thein vivo response of the heart to hypothermiaand the in vitro metabolic response of certainenzyme systems to various temperatures. Itwas found that in vivo responses as assayedby changes in body temperature, heart rate,and electrocardiographic measurements wasquite similar to those of larger animals. Theonly exception insofar as the heart was con-cerned was the very low incidence of ventric-ular fibrillation. In the in vitro metabolicstudies it was observed in Arrhenius-van'tHoff plots that ventricular hypothermia wasassociated with a higher temperature-velocityconstant below a temperature of 20 to 21 C.than above it. This was characteristic of tri-carboxylic-acid cycle substrate oxidations aswell as of endogenous respiration. The rela-tionship of cardiac function to myocardialmetabolism in hypothermia and the signifi-cance of shifts in the temperature-velocityconstant are discussed.

SUMMARIO IN INTERLINGUA

Esseva effectuate, in rattos, studios experi-mental pro establir le natura del responsa car-diac in vivo a hypothermia e del responsa me-tabolic in vitro de certe systemas de enzymasa varie temperaturas non-natural. Esseva no-tate que le responses cardiac in vivo, mani-feste in alterations del temperatura corporee,del frequentia del corde, e de mesurationeselectrocardiographic, es satis simile in rattose in plus grande animales. Le sol exceptionesseva le bassissime incidentia de fibrillationventricular in le rattos. In le studios meta-bolic in vitro, il esseva notate in diagrammasde Arrhenius-van't Hoff que hypothermia es-seva associate con un plus alte constant* detemperatura-velocitate a temperaturas infra20 a 21 C que a temperaturas plus alte. Istoesseva characteristic de oxydationes a sub-strate de cyclo de acido tricarboxylic e etiamde respiration endogene. Es discutite le re-

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778 HAXXOX

lation inter le function cardiac e le rnetabo-lismo myocardial in hypothermia e le signif-ication de variationes del constante de tem-peratura-velocitate.

REFERENCES1. HANNON, J. P., AND COVIXO, B. G.: Effect of

hypothermia on the cellular respiration ofventricular tissue. Am. J. Phyniol. 192: 121,1958.

2. UMBREIT, W. W., BURRIS, R. H., AND STAUF-FER, J. F.: Manometric Techniques. Minne-apolis, Burgess Publishing Co., 1957.

3. CRISMON, J. M.: Effect of hypothermia onthe heart rate, arterial pressure and theelectrocardiogram of the rat. Arch. Int.Med. 74: 235, 1944.

4. KAYSER, C.: Physiological aspects of hypo-thermia. Ann. Rev. Physiol. 19: S3, 1957.

5. —, RIETSCH, M. L., AND LUCOT, M. A.: Lesechanges respiratoires et la frequence car-diaque des hibernants au cours du reveil deleur sommeil hivernal; recherches physiolo-giques sur l'increment thermique critique.Arch. sci. physiol. 8: 155, 1954.

6. MEDA, E.: Richerche sull' attivita' cardiacanella cavia durante il raffreddamento cor-poreo. Med. sper. 23: 3, 1953.

7. COOKSO.V, B. A., AND DIPALMA, J. R.: Effect

of qunrternary nitrogen compounds on theS. A. node in profound hypothermia. Am.J. Physiol. 188: 274, 1957.

S. COVINO, B. G., CHARLESON, D. A., ANDD'AMATO, H. E.: Ventricular fibrillation inthe hypothermic dog. Am. J. Physiol. 178:14S, 1954.

9. JOHNSON, F. H., EYRING, H., AND POLISSAR,M. J.: The Kinetic Basis of Molecular Biol-ogy. Xew York, John Wiley & Sons, Inc.,1954.

10. BURTON, A. C.: The basis of the principal ofmaster reaction in biology. J. Cell, andComp. Physiol. 9: 1, 1936.

11. CROZIER, AV. J.: On biological oxidations asa function of temperature. J. Gen. Physiol.7:189,1925.

12. HADIDIAN, Z., AND HOAGLAND, H.: Chemicalpacemakers. Part I. Catalytic brain iron.Part II. Activation energies of chemicalpacemakers. J. Gen. Physiol. 23: 81, 1940..

13. BELEHRADEK, J.: Temperature and the rateof enzyme action. Nature 173: 70, 1954.

14. KAVANAU, J. L.: Enzyme kinetics and the rateof biological processes. J. Gen. Physiol. 34:193, 1950.

15. MORALES, M. F. : A note on limiting reactionsand temperature coefficients. J. Cell, andComp. Physiol. 30: 303, 1947.

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JOHN P. HANNONOxidations of the Rat

Effect of Temperature on the Heart Rate, Electrocardiogram and Certain Myocardial

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1958 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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