distribution of norepinephrine in the failing bovine...

Distribution of Norepinephrinein the Failing Bovine Heart

CORRELATION OF CHEMICAL ANALYSIS

AND FLUORESCENCE MICROSCOPY

By John H. K. Vogel, M.D., David Jacobowitz, Ph.D.,

and Charles A. Chidsey, M.D.

ABSTRACTStudies were carried out during experimental heart failure induced in calves

to compare the depletion of cardiac norepinephrine in the failing heart to thehistochemical appearance of the adrenergic innervation in this tissue. A cor-relation has been established between the reduction of norepinephrine concen-tration and the changes in the distribution of the adrenergic neurotransmitterin the tissues. Absence of fluorescence in terminal varicose fibers in closeassociation with cardiac muscle cells was characteristic of the failing heart.Little or no change occurred in preterminal fibers or in terminal fibers inconnective tissue septa or around blood vessels. In two steers, recovery fromheart failure was shown to be associated with a virtual restoration of thenorepinephrine concentration and of the histochemical appearance of theadrenergic nerve distribution in 28 days or less. These findings suggest thata reversible abnormality, possibly of neurotransmitter storage or synthesis, orboth, is induced in the terminal portion of the cardiac adrenergic innervationas the result of the development of the failure state.

ADDITIONAL KEY WORDS chromaffin cells mast cellsadrenergic nerves fluorescent histochemistry myocardial innervationcalves

• A marked reduction in norepinephrineconcentration has been observed in the failingheart of both man (1) and experimental ani-mals (2). Previous biochemical and pharma-cological studies have suggested that thisreduction was associated with a significantfunctional impairment of the cardiac sympa-thetic nerves, involving both storage (3) andsynthesis (4) of the neural transmitter sub-

From the Divisions of Cardiology and Clinical Phar-macology, Department of Medicine, University ofColorado Medical Center, Denver, Colorado, and De-partment of Pharmacology, University of Pennsylvania,School of Medicine, Philadelphia, Pennsylvania.

This investigation was supported in part by GrantAHA 66728 from the American Heart Association andby U. S. Public Health Service Research GrantsHE-09932, 5TO1 HE-05390, and 1RO1 NB-06707-02.

Dr. Chidsey is a Burroughs-Wellcome Scholar inClinical Pharmacology, and Dr. Jacobowitz is a re-cipient of a Research Career Program Award, 5-K 3-NB-13, 935-02, from the National Institutes of Health.

Accepted for publication November 1, 1968.

stance. The importance of this biochemicalabnormality has been demonstrated by theobserved depression of the response to stimu-lation of the postganglionic nerves supplyingthe failing heart (5). However, in spite ofthese studies demonstrating the functionalsignificance of this abnormality, the mecha-nisms which may be responsible for its de-velopment remain largely unexplained.

The present study was undertaken to de-fine the microanatomical distribution of theadrenergic nerves in control, hypertrophied,and failing hearts and to relate these observa-tions with chemical measurements of the totaladrenergic neurotransmitter contained withinthe myocardial tissue. In addition, studieswere carried out in hearts following recoveryfrom the failure state. For this purpose abovine experimental heart failure preparationwas used (6). In this study a correlation hasbeen observed between the depletion of total

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72 VOGEL, JACOBOWITZ, CHIDSEY

tissue norepinephrine determined chemicallyand the reduction of catecholamine withinadrenergic nerves in the failing heart. Further-more, the microanatomical abnormality ofthe cardiac adrenergic nerves appeared to beoriented principally around terminal nerveendings, suggesting a localized dysfunctionof the nerves in their terminal varicose por-tions. When animals were studied after re-covery from heart failure, both the micro-anatomical and the chemical abnormalitieshad returned virtually to the control condi-tion.

MethodsRight pulmonary artery ligation was performed

within 48 hours after birth in 17 calves whichwere born at 5,280 ft. This operation resulted inprogressive pulmonary hypertension with rightventricular hypertrophy and subsequent develop-ment of heart failure (6). The pulmonary arteryligations were performed through a right thora-cotomy at the level of the fourth intercostal spaceusing halothane1 and nitrous oxide anesthesia.The branches of the right pulmonary artery tothe upper, middle, and lower lobes were ligatedindividually, care being taken to avoid any ma-nipulation of the great vessels or the pericardium.The animals recovered from the operative proce-dure, and at a later time hemodynamic studieswere carried out, both before and after thedevelopment of heart failure. Cardiac catheteri-zation was performed via an external jugularvein with the animals standing awake and un-sedated (6). Ten animals were studied prior tofailure; four at 3 days, three at 10 days, andthree at 28 days. Three calves were studiedwithin 1 week after the onset of failure (earlyheart failure); four calves were studied afterhaving been in failure for more than 1 week (lateheart failure). Heart failure was considered tobe present when persistent distention of theexternal jugular veins was observed. This initialmanifestation of failure was followed by pro-gressive fluid accumulation in the animals leadingto obvious ascites and distention of the brisket.

We have previously shown that left pulmonaryartery ligation in calves at sea level does notresult in progressive pulmonary hypertension (6).However, when such animals are brought toDenver (5,280 ft) progressive pulmonary hyper-tension and heart failure will occur in theseanimals. This hemodynamic sequence can be

'Kindly supplied as Fluothane by Ayerst Labora-tories.

reversed by returning the hypertensive animalwith right heart failure to sea level. Using thispreparation we were able to obtain observationsin 36-month-old steers both in severe heartfailure and following recovery from late heartfailure effected by returning the animals withheart failure to sea level. Four steers that hadfailed to develop pulmonary hypertension fol-lowing left pulmonary artery ligations at sealevel were transported to Denver where theydeveloped progressive pulmonary hypertensionand congestive heart failure. Two of these werekilled after they had demonstrated evidence offailure for 1 month. Two others, which had alsobeen in failure for 1 month, were returned to sealevel for 10 and 28 days, respectively, and thenkilled. Two unoperated animals of a comparableage were killed as controls.

Following the hemodynamic measurements, theanimals were killed and the hearts removed forchemical and histochemical determinations. Thehearts were placed on crushed ice for sectioning.Tissues for norepinephrine determination werefrozen on dry ice and later assayed by a fluoro-metric method (7, 8). Tissues for fluorescencemicroscopy were rapidly removed from eachcardiac chamber and immediately frozen in iso-pentane cooled by liquid nitrogen. Subsequent-ly, these specimens were freeze-dried at —30°C,treated with stock paraformaldehyde vapor at 80°C for 1 hour, embedded in paraffin under vacuum,and sectioned at 14/j, for fluorescence microscopyaccording to the method of Falck and Hillarp(9-11). The specificity of this method has beendemonstrated for norepinephrine, epinephrine,and dopamine, which give a green fluorescence,as distinct from 5-hydroxytryptamine, which givesa yellow fluorescence (10). Although quantifica-tion is not possible with this technique, one canroughly estimate whether an experimental pro-cedure has caused an increase, decrease, or nochange in neuronal content of monoamines. Forpurposes of quantification, the standard chemicalassays were used. After removal of the myo-cardial specimens, the hearts were stripped ofatria, fat, great vessels, and valve tissue, and theindividual weights of the free wall of the rightventricle and left ventricle with septum weredetermined.

ResultsCHEMICAL STUDIES OF NOREPINEPHRINE

Normal Hearts.—The concentrations of nor-epinephrine in the heart were similar in thecontrol animals at ages varying from birth to18 months. Little change was found in thenorepinephrine concentration in these tissuesbetween birth and 18 months, but the nor-

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NOREPINEPHRINE IN THE FAILING HEART 73

Norvial Bovine

Age

1 clay

2 weeks

4 weeks

8 weeks

MEAN*

18 months

MEAN

TABLE 1Cardiac Norepinephrine (Vg/

Rightatrium

1.360.79

1.812.36

1.121.43

1.301.591.44

1.47

1.131.76

1.44

TOTAL MEAN 1.46

SE 0.13

Rightventricle

1.470.94

1.802.08

1.251.76

1.121.381.69

1.50

1.460.87

1.16

1.440.11

Leftatrium

1.990.76

1.212.90

1.081.31

1.501.711.63

1.57

1.261.44

1.35

1.530.17

/g)

Leftventricle

1.481.00

0.881.19

0.971.28

0.941.291.44

1.16

1.110.64

0.88

1.110.08

*Mean of all measurements up to and includingthose at 8 weeks.

epinephrine concentration was lower in theventricles of the 36-month-old animals (Tables1 and 2). Except in the animals killed im-mediately after birth, right ventricular nor-epinephrine tended to be higher than thevalues determined in the left ventricle.

Hypertrophy.— Progressive hypertrophy ofthe right ventricle was observed in thoseanimals in which right ventricular hyperten-sion had been induced, both before and afterthe development of failure. The right ven-tricular portion of the total ventricular weight

RV-=rXl00) was increased in the animals

before the onset of failure from a controlvalue of 33 ± 0.7% (average right ventricularpressure 48/5 mm Hg) to 38 ± 2.3% at 10 days(right ventricular pressure 90/7 mm Hg), and42±2.3% at 28 days (right ventricular pres-sure 133/12 mm Hg). Three days followingsurgery it was 32 ± 1.2% (right ventricularpressure 78/8 mm Hg), which was not sig-nificantly different from the controls. Therewas only a minimal alteration in cardiacnorepinephrine concentration in these hyper-trophied nonfailing hearts (Fig. 1). The right

ventricular norepinephrine concentration wasslightly reduced at 10 days, averaging 0.75/ig/g. However, by 28 days this value hadincreased to nearly normal levels, averaging1.16 /*g/g.

Heart Failure.--In the animals with failure,both early and late, a persistent increase inthe right ventricular percentage of total ven-tricular weight was noted, averaging 48 ±2.7% in early failure (right ventricular pres-sure 94/22 mm Hg) and 52 ±3% in latefailure (-right ventricular pressure 104/30 mmHg). In both stages of failure, cardiac nor-epinephrine concentration was decreased(Fig. 1). There was an obviously greaterdecrease in the right ventricular and rightatrial values than in those of the left atriumand ventricle. A more striking depletion ofnorepinephrine was observed in the late stageof failure in the right ventricle; mean valueswere 0.11 /tg/g in late failure compared to0.38 jU,g/g in early failure. Similar differencesbetween these two stages of failure wereobserved in the other cardiac chambers.

Recovery from Failure.—The norepineph-rine concentrations in the hearts of two ofthe 36-month-old animals, killed after demon-strating evidence of failure for 1 month, weregreatly reduced (Table 2) compared to thevalues observed in the two control animals.In the two animals with failure, in which

TABLE 2

Cardiac Norepinephrine (fig/g) in Thirty-Six-Month-Old Steers

Rightatrium

Rightventricle

Leftatrium

Leftventricle

No. 1No. 2

MEAN

No. 1No. 2

MEAN

10 days28 days

MEAN

Control1.261.09

1.18

Steers in0.320.34

0.33

Steers after0.431.41

0.92

Steers0.580.60

0.59

Failure0.160.12

0.14

Recovery0.330.52

0.42

1.481.19

1.34

0.78

0.78

1.231.41

1.32

0.580.86

0.72

0.400.29

0.34

0.650.44

0.54



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2.0

1.0 iRA

rh

3 10 28

2.0n

1.0

LA

C 3 10 28

2.0

1.0-

RV

i3 10 28

2.0n

1.0-

LV

E LCHF

FIGURE 1

I rH4i

3 10 28

IE LCHF

Mean values and SEM (indicated by bar) for cardiac norepinephrine concentrations (ordinate)in control calves (C); at 3, 10, and 28 days after right pulmonary artery ligation; and in early(E) and late (L) congestive heart failure (CHF). RA = right atrium, LA = left atrium, RV =right ventricle, and LV = left ventricle.

recovery from heart failure had been effectedby return to sea level for 10 and 28 days,respectively, the cardiac norepinephrine wasclearly elevated above the levels observed infailure, but was not quite as high as the levelsobserved in the hearts of the two control36-month-old steers (Table 2). The steer thattook 28 days to recover was essentially nor-mal.

HISTOCHEMICAL OBSERVATIONS

Normal and Hypertrophied Calf Hearts

The histochemical appearance of the adre-nergic innervation was essentially the same inboth normal and hypertrophied calf hearts.All catecholamine-containing fibers had agreen fluorescence specifically characteristicof norepinephrine and epinephrine.

Atria.—Both atria contain a large numberof varicose nerve fibers with an intensefluorescence (Fig. 2). Fine varicose nerveterminals were seen in intimate contact withcardiac muscle fibers. In addition, larger pre-terminal nerve trunks containing varicosefibers were present in connective tissue re-gions within the heart and in close proximity

to blood vessels (Fig. 3, A). The smoothmuscle of the coronary arteries was surround-ed by a dense network of adrenergic nerveterminals (Fig. 2, B). There was no apparentdifference between the nerves to the rightand left atria. However, the A-V nodal re-gion within the interatrial septum containeda dense network of catecholamine fluorescentnerves (Fig. 2, C). In addition, many clustersof intense green-yellow fluorescent cells wereobserved in the interatrial septum close tononfluorescent (cholinergic) ganglion cells.These fluorescent cells strongly resemble thechromaffin cells of the adrenal medulla insize, shape, color, and intensity of fluores-cence. In the atria, they are primarily in theinteratrial septum closely associated withganglion cells and blood vessels. They areusually found in clusters of approximately5 to 15 cells.

Ventricles.— The ventricles contain a largenumber of fluorescent nerve fibers similar indensity and distribution to those describedabove for the atria (Figs. 2, D and 3, A). Inboth ventricles the bundle branches of theventricular conducting system can be readily



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FIGURE 2

A: Left atrium, normal calf. Adrenergic varicose nerve fibers are observed coursing over thecardiac muscle cells. Smooth autofluorescent elastic fibers are also observed (X170). B: Coronaryartery, normal calf left atrium. Adrenergic nerve fibers following the course of a branchingcoronary artery. Note dimly fluorescent preterminal nerve trunk (arrow) (xl70). C: Calf A-Vnode. Dense innervation with adrenergic nerves (X 170). D: Right ventricle, normal calf.Adrenergic fibers are similar in distribution as in the atria (X 170).

identified using the fluorescence microscope.This tissue is essentially devoid of the diffuse


faint-green autofluorescence observed in thecardiac muscle. However, autofluorescent


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FIGURE 3

A: Large preterminal nerve trunk in right ventricle. Note varicose fibers within the trunk (X170).B: A bundle branch of the atrioventricular conducting system (AVC) of the calf. Note adrenergicfibers coursing within the surrounding connective tissue sheath. Note also fluorescent mast cells(arrow) (x 170). C: Nonfluorescent cholinergic ganglion cells (G) in the epicardium of theleft ventricle of the calf. Fluorescent terminal varicosities (white arrows) appear to make contactwith the ganglion cells. A terminal nerve fiber (black arrow) is seen emanating from a nervetrunk below (x260). D: Green-yellow fluorescent chromaffin cells in the epicardial region ofthe left ventricle of a calf in heart failure. These cells were close to a ganglion (x660).



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FIGURE 4

Calf, early heart failure (X170). A: Left atrium. Similar to normal atrium. B: Right atrium.Moderate number of nerve terminals compared to left atrium. Normal appearing preterminalnerve trunks (FT) in connective tissue (CT) region. Note fluorescent mast cells (arrows). C:Left ventricle. Similar to normal ventricle. D: Right ventricle. Markedly reduced content ofnerve terminals with diminished intensity of fluorescence. Preterminal nerves are present as innormal ventricle.

orange granules are present in some of thecells of this tissue. The identity of these


granules is unknown. The conduction bundlesare surrounded by green autofluorescent con-


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FIGURE 5

Calf, late heart failure (xl70). A: Left atrium. Reduced number and intensity of fluorescenceof terminal nerve fibers. Two blood vessels (arrows) with more intensely fluorescent nerve fibers.B: Right atrium. Markedly reduced number of nerve terminals. Blood vessels (arrows) withintense fluorescent nerve fibers. Note normal-appearing mast cells. C: Left ventricle. Markedreduction in number and intensity of fluorescence of nerve terminals. Fibers in connective tissuespaces appear normal (arrows). D: Right ventricle. Similar to left ventricle. Note large smoothfluorescent preterminal nerve trunk (arrow) in connective tissue septa.



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.'IFIGURE 6

Steer, recovery from heart failure, 28 days. All chambers essentially normal (X170). A: Leftatrium. B: Right atrium. C: Left ventricle. D: Right ventricle.

nective tissue containing elastic fibers. Fre-quently, adrenergic fibers can be seen cours-ing within the surrounding connective tissue


sheath, but no nerve fibers ramify or terminateupon the individual cells of the bundlebranches of the conducting system (Fig. 3,


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B). In one calf, nonfluorescent ganglion cellswere observed in the epicardial connectivetissue of the left ventricle (Fig. 3, C). Severalof the cells appeared to be innervated byadrenergic nerve terminals. In another calf(with heart failure, described below) non-fluorescent ganglion cells were found in theepicardial region of the left ventricle. Inclose proximity to these was a large clusterof green-yellow fluorescent chromaffin cells(Fig. 3, D). In addition, a large, smooth,green fluorescent preterminal nerve trunk wasobserved in the confines of the ganglion.

In both atria and ventricles an abundantnumber of intense green fluorescent mast cellswere present between the muscle fascicleswithin the connective tissue (Figs. 3, B; 4, B;5, B and C; and 6, B). Green fluorescentdistinct granules were usually observed sur-rounding a nonfluorescent nucleus.

Heart Failure

Early Failure.—In the left atrium the num-ber of fluorescent nerve fibers and the inten-sity of their fluorescence were essentiallysimilar to those of the normal calf atrium(Fig. 4, A). The right atrium contained onlya moderate number of fluorescent nerve ter-minals, i.e., much fewer than the left atrium(Fig. 4, B). The intensity of fluorescence wasreduced in many of the fibers. However, thelarge preterminal nerve trunks in the con-nective tissue regions appeared to be normalin number and intensity of fluorescence. Inthe left ventricle the content of fluorescentnerves and intensity of their fluorescence ap-peared to be normal (Fig. 4, C). The rightventricle contained a markedly reduced con-tent of fluorescent terminals whose intensityof fluorescence was diminished (Fig. 4, D).Here too, preterminal nerve trunks present inthe intermuscular connective tissue appearednormal. Green fluorescent mast cells werepresent in all the chambers of the heart. Nounusual changes in the catecholamine con-tent of these cells were noted.

Late Failure.—A moderate-to-marked re-duction of the fluorescent terminals and theintensity of their fluorescence was seen in the

left atrium (Fig. 5, A). In all specimens,those nerves contained within the connectivetissue septa between the muscle bundles wereusually much more intensely fluorescent thanthose nerve terminals within close proximityto the muscle cells. Large intense fluorescentpreterminal nerve trunks were present primar-ily in connective tissue regions. Nerve fibersof normal intensity of fluorescence were ob-served surrounding the coronary arteries be-tween the tunica adventitia and tunica media.In the right atrium the number of fluorescentfibers and the intensity of their fluorescencewas very low (Fig. 5, B). Some large pre-terminal nerve trunks containing varicosefibers were observed in connective tissue re-gions and in close proximity to blood vessels.In the left ventricle a marked reduction inthe number of fluorescent nerve fibers andthe intensity of their fluorescence was ob-served (Fig. 5, C). Some nerves with normalintensity of fluorescence were present primari-ly in the connective tissue spaces. Bloodvessels were generally innervated with nervefibers having normal fluorescence. Large,smooth, fluorescent preterminal nerve trunkswere present in connective tissue regions andadjacent to blood vessels. The A-V conductingbundle branches showed no unusual histo-logical changes. The orange fluorescent gran-ules were still present and unaltered in con-tent or intensity of fluorescence. Terminalvaricose fibers were present in the connectivetissue surrounding the bundle branches. Inthe right ventricle the number of fluorescentnerve fibers and the intensity of their fluores-cence was markedly reduced (Fig. 5, D).However, the blood vessels contained manyintensely fluorescent fibers surrounding thetunica media. Large, smooth, preterminalnerve trunks were present primarily in theconnective tissue septa and these demonstrat-ed fluorescence of moderate intensity.

Studies Carried Out After Recoveryfrom Heart Failure in Steers

The histochemical picture of the myocar-dium of control steers was similar to that ob-served in control calves except that therewere fewer flourescent nerve fibers in the ven-



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tricles. In addition, there were fewer fluores-cent preterminal nerve trunks.

The adrenergic nerves in the heart of thesteer studied 10 days after beginning recoveryfrom failure demonstrated a substantial in-crease in both fluorescence intensity and num-ber of fluorescent terminal varicosities in theright atrium and ventricle, whereas the leftatrium and ventricle appeared essentiallynormal. In the steer studied 28 days afterbeginning recovery from failure, the adre-nergic nerves in all four chambers appearedessentially normal (Fig. 6).

Discussion

In these studies a comparison has beenmade between alterations in myocardial nor-epinephrine concentration determined chemi-cally and changes in the fluorescence ofadrenergic nerves during the developmentof heart failure in calves with experimentallyinduced pulmonary hypertension. Our find-ings have demonstrated a correlation betweenthe depletion of myocardial norepinephrineand changes in catecholamine fluorescence(9-11). Little or no chemical or histochemicalchange was seen in the hypertrophied heartprior to the onset of hemodynamic evidenceof failure. These findings are in contrast toobservations of a striking depletion of cardiacnorepinephrine in the nonfailing hypertro-phied ventricle reported by Spann et al. (12).However, in their studies, marked right ven-tricular hypertension was produced acutelyby surgical constriction of the main pulmonaryartery, and it is entirely possible that thisprocedure may have led to ischemic injuryof the right ventricular myocardium (13) andof the sympathetic innervation of the heart.With the onset of failure, we observed botha depletion of chemically determined nor-epinephrine and a marked reduction in thenumber of fluorescent terminal adrenergicnerves in close proximity to the cardiac mus-cle cells with an absence of the fine varicosi-ties characteristic of the adrenergic groundplexus. However, these varicose terminalnerve fibers were unchanged in intermuscularseptal areas and also in areas surrounding


blood vessels. In early failure the changes inboth norepinephrine concentration andadrenergic nerve distribution were less strik-ing than in the later stages of failure, sug-gesting that failure of cardiac performanceprecedes, and is not the result of, norepi-nephrine depletion in the heart. In early fail-ure fluorescent terminal fibers were decreasedin some muscle bundles, whereas in latefailure there was a greater reduction in allmuscle bundles. The absence of fluorescentneurons does not necessarily indicate an ana-tomic loss of neural element, since it can alsobe explained by metabolic dysfunction of theneuron, with subsequent diminution of storednorepinephrine within the adrenergic ter-minal.

Mast cells with an intense green fluores-cence were observed in all chambers of thesehearts. There was no obvious alteration in theintensity of fluorescence or number of thesecells in any stage of heart failure examinedin this study. Previous investigations of mastcells in ruminant tissue have demonstratedthat the distribution of these cells corre-sponds with the occurrence of dopamine (14).The functional role of these mast cells in theheart is unclear. It is recognized that dopa-mine is an effective beta-receptor agonist withinotropic properties (15). The absence ofnotable structural abnormality of these cellssuggests that the mechanisms responsible forthe loss of fluorescence in the neuronal tissuedo not similarly affect these dopamine con-taining cells.

The calf and steer are one of the few speciesthat has parasympathetic ganglion cells inboth the ventricles and atria. Both adrener-gic terminal varicosities and clusters ofchromaffin cells were present in close proximi-ty to ganglion cells. This is consistent withobservations in several other species (16).The release of norepinephrine from theseendings and chromaffin cells could result in adepression of ganglionic transmission andtherefore serve as a possible modulatory in-fluence on cholinergic transmission throughthe ganglion. It has been well documentedthat catecholamines are capable of depress-


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ing ganglionic impulse transmission (17-19).It is of interest that although nodal tissuehas a dense adrenergic innervation, the bun-dle branches of the atrioventricular conduct-ing system were essentially devoid of sympa-thetic nerves. However, a considerablenumber of nerves are present within theconnective tissue immediately surroundingthe bundle branches within the ventricles.This arrangement is reminiscent of sympa-thetic nerves within the tunica adventitia ofarteries in close apposition to the muscle (tuni-ca media) and suggests a possible influenceof adrenergic nerves on the atrioventricularconducting system. No apparent change inthe catecholamine content of this adrenergicinnervation was observed in the failing heart.It was somewhat unexpected from previous-ly reported observations (20) that the cardiacadrenergic innervation was essentially com-pletely established in our newborn calves,showing no further development in the earlyperiod of body growth.

The changes in the adrenergic innervationof the failing heart, which were noted pre-dominantly in the terminal varicose fibers inclose approximation to muscle bundles, mayindicate that these fibers were influenced insome manner by an alteration taking placeat the region of close association between thenerve terminal and the cardiac muscle cell.This histochemical picture is similar to thatobserved in iris tissue removed from animalspretreated with reserpine in which the neuro-transmitter store was partially repleted byadministration of norepinephrine. In thesetissues there was a marked reduction influorescence of terminal varicosities, with less-er involvement of the preterminal nerves(21). The alteration in these reserpinizedtissues was presumed to be due to inter-ference with an ATP-Mg+ +-dependent mech-anism for the binding of amines. However,there is some question whether a deficit inmyocardial ATP is present in the failingheart. Thus, surgical specimens obtained fromfailing human hearts reveal no change inATP or creatine phosphate concentrationsand mitochondria prepared from failing hu-

man hearts have normal oxidative phosphoryl-ation (22). Although a minor reduction increatine phosphate has been observed in ex-perimental heart failure, other variables re-lating to energy production are normal (23).However, in spite of the lack of evidencesuggesting a defect in energy production inthe tissue as a whole, localized areas ofhypoxia may occur in the myocardium as aresult of hypertrophy and dilation of thefailing ventricle (24). Metabolic derange-ments associated with such hypoxic areasmay lead to a substantial regional alterationof neural function. One possible metabolicderangement might involve the productionof an abnormal intermediate in the biosyn-thesis of norepinephrine. Thus, 6-hydroxy-dopamine has been shown both to depletenorepinephrine from adrenergic nerves andactually to alter neural structure (25).

In these studies we have demonstrated notonly that a marked alteration in the adre-nergic innervation occurs in the failing myo-cardium, but also that with recovery fromheart failure reconstitution occurs. Substantialreconstitution of the normal pattern of adre-nergic nerves occurred as early as 10 days afterreturning the animals to an environmentwhere cardiac compensation was again possi-ble. We cannot determine from these ob-servations whether the process of recoveryinvolved a restoration of anatomically absentnerve terminations in the substance of themuscle bundles or a recovery of intraneuron-al function necessary for the storage and syn-thesis of a normal complement of neurotrans-mitter substance in the terminations. It istraditionally accepted that nerve regrowthoccurs at the rate of approximately 1 mm/day (26). This value was derived from mye-linated neurons in situ. Less precise informa-tion is available about the rate of regrowth ofnonmyelinated adrenergic neurons (postgan-glionic nerves), the only studies being thoserelating to the reinnervation of the nictitatingmembrane and the surgically denervated heart(27, 28). Although these observations suggesta slower rate of regrowth of postganglionicnerves, even this could account for the re-

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establishment of the normal microanatomicalpattern in the heart which we have studiedfollowing recovery from the failure state. Thedistances involved in this process are merelythose between the preterminal nerves in theintermuscular septa and the terminal varicoseelements within the muscle bundle, a distanceprobably not exceeding 100 to 200/A. Regard-less of whether regrowth of neurons or re-constitution of neuronal function is responsiblefor the present findings, it is clear that cardiacadrenergic neurotransmitter depletion is rela-tively rapidly reversible on recovery fromheart failure.

Acknowledgments

The authors wish to thank Gail Jamieson, DorothyWalker, and Ruth Eussner for their excellent technicalassistance; J. D. McCrady, D.V.M., of Texas A & M,College Station, Texas, for following the steers at sealevel; Mr. Harry Mills, Supervisor of the animalchambers, for his excellent animal care; and MarilynLeek for typing the manuscript.

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AND MASON, D. T.: Myocardial norepineph-rine concentrations in man: Effects of reserpineand congestive heart failure. New Engl. J.Med. 269: 653, 1963.

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E. H., SPANN, J. F., JR., AND BRAUNWALD,

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G., ROSENBERG, H., JAMIESON, G., AND MC-

CRADY, J. D.: Effects of mild chronic hypoxiaon the pulmonary circulation in calves with re-active pulmonary hypertension. CirculationRes. 21: 661, 1967.


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proved technique for fluorometric estimationof catecholamines. Acta Physiol. Scand. 51:348, 1961.

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JOHN H. VOGEL, DAVID JACOBOWITZ and CHARLES A. CHIDSEYAnalysis and Fluorescence Microscopy

Distribution of Norepinephrine in the Failing Bovine Heart: Correlation of Chemical

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