Postgraduate Medical Journal (May 1975) 51, 334-339.

New approach in studying cardiac muscle cell injury*

G. RONAt M. BOUTETM.D., Ph.D M.D., Ph.D.

I. HUjTTNERM.D., Ph.D.

Pathology Institute, McGill University, Montreal, Canada*

SummaryA new approach for investigating the mechanisms andevolution of cardiac muscle cell injury was presentedby applying fine structural extracellular protein tracersin the catecholamine-induced cardiac muscle cellinjury model. The results with these diffusion tracersindicated striking functional changes at the level ofcoronary microcirculation followed by early per-meability alteration of cardiac muscle cell membranes,thus suggesting the role of these factors in myocardialchanges which develop without narrowing or obstruc-tion of coronary arteries.

DURING the past 15 years, investigations of our re-search group concerned themselves with the studyof myocardial reaction and adaptation to insultunder various experimental conditions (Rona, 1971).Recently ultrastructural and functional correlativestudies carried out with catecholamines with con-tinuous low dose (4-6 tLg/100 g body w/min) infusionshowed that isoproterenol and norepinephrine pro-duced irreversible cardiac muscle cell lesion. On onehand, the cardiac muscle cell lesions produced byvarious catecholamines have certain similar mor-phological features which were related to theircommon metabolic effect (mitochondrial respiration,glycogenolysis, fatty acid mobilization, electrolyteshift). On the other hand, their diverse effect on themyofilaments and the interstitial reaction indicateddifferences in the pathogenesis of the myocardiallesions elicited by the two catecholamines. Whereasisoproterenol induced primarily myofilament hyper-contraction and contraction bands, lytic lesions pre-dominated in the norepinephrine-induced cardiacmuscle cell changes. Moreover, rats treated withnorepinephrine showed early and marked inter-stitial oedema, whereas in rats treated with iso-proterenol, the evolution of oedema was delayed byhours (Boutet, Hiittner and Rona, 1973, 1974).

* Grant No. MT-3635.

t Department of Pathology, McGill University, Montreal.

Some of the structural differences were related tothe diverse circulatory effect of the two catechol-amines. Isoproterenol is a depressor amine, whilenorepinephrine is a strong pressor agent. Whereasseveral investigations dealt with the role of vascularfactors in the pathogenesis of myocardial lesionsproduced by catecholamines, little attention was paidto the functional effect of these agents on the localcoronary microcirculation. Extracellular fine struc-tural protein tracers can be utilized with advantageto investigate how catecholamines affect the func-tional state of coronary capillaries. In our labora-tory, peroxidase and ferritin were used for suchmicrocirculatory studies (Huttner, Boutet and More,1973a, b; Huttner et al., 1973). Peroxidase moleculesthat have a diameter of approx. 50A and molecularweight of 40,000 traverse the endothelial barrierthrough intercellular clefts and by plasmalemmalvesicles. Ferritin which has a diameter of approx.1 IOA and molecular weight of 500,000 is transportedexclusively by plasmalemmal vesicles of the coronarycapillary endothelium.

Catecholamines were infused under ether anaes-thesia in a dose of 4-6 Fg/100 g body w/min, whilethe systemic blood pressure was recorded on apolygraph. Saline-treated animals were used ascontrols for tracer experiment. The observation ofdelayed phase of interstitial oedema in the isopro-terenol experiment necessitated the introduction ofanother experimental group in which isoproterenolwas given subcutaneously in a dose of 8-5 mg/100 gbody wt and the tracers were injected at the time ofthe delayed oedema, i.e. 90 min following isopro-terenol injection. Diffusion tracers were injected atvarious time intervals before sacrificing the animalsby perfusion of aldehyde fixative through thecoronary capillaries. Horseradish peroxidase wasinjected in a dose of 10 mg/100 g body wt, whereasthe dose of ferritin was 100 mg/bOO g body wt.

Figure 1 shows the polygraph records of carotidartery pressure from control, as well as norepine-phrine, and isoproterenol-infused rats. Note that

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Study of cardiac muscle cell injury 335

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336 G. Rona, M. Boutet and I. Huttner

the records read from right to left. Parallel arrowsindicate the peroxidase circulation time which, inthis experiment, was 60 sec. Similar recordings areobtained with ferritin. Continuous intravenous in-fusion of norepinephrine produces a rise of bloodpressure from the control 115± 15 mmHg to190±20 mmHg. This high blood pressure level re-mained constant during the circulation time ofperoxidase. Following isoproterenol infusion thereis an immediate drop of arterial blood pressure to70±15 mmHg. The ultrastructure of endothelialcells was comparable with that of controls in animalstreated with catecholamines under the selectedexperimental conditions. Studies with protein tracersrevealed, however, striking functional changes atthe level of microcirculation.

Figure 2 presents light microscopic picture ofright ventricular myocardium from the selected

experimental groups. Upper set of pictures madeafter 60 sec, and the lower pictures after 6 mincirculation time of the tracer. As compared with thenegative control (a), the myocardial interstitiumshows diffuse staining by reaction product followingNE-induced high blood pressure (b). At 6 min thereis uniform distribution of the tracer in control myo-cardial interstitium (c). The tracer is, however,absent following isoproterenol induced low bloodpressure (d).

Figure 3 presents the same experimental arrange-ment and fine structural localization of peroxidasereaction product. In control, the tracer is localizedin the lumen of the capillary, in lumenal plasmalem-mal vesicles and in the lumenal side of interendo-thelial clefts, but the myocardial interstitium isnegative for peroxidase (a). In contrast to the nega-tive control, intensive reaction is obtained all along

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Study of cardiac muscle cell injury 337

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interendothelial clefts, ablumenal vesicles and in themyocardial interstitium following identical 60 secperoxidase circulation time in rat with norepine-phrine-induced high blood pressure (b). Six minutesfollowing peroxidase injection, peroxidase reactionproduct is already localized in the interstitium ofcontrols (c). Following identical 6 min circulationtime from rat with isoproterenol-induced low bloodpressure, peroxidase labels some large endothelialvesicles but no reaction product is discernible in themyocardial interstitium, indicating uptake but notransendothelial transfer of the tracer (d).

Figure 4 shows that as early as 10 min followingnorepinephrine infusion the sarcoplasm of somecardiac muscle cells is also positive for peroxidase,indicating that the sarcoplasmic membrane of thesecells became permeable to the tracer. Note thatperoxidase within the sarcoplasm is deposited upon

and bound selectively to myofilaments. These cellsotherwise present normal ultrastructure. The samefindings were present in isoproterenol studies in therecuperation phase when the arterial blood pressurereturned towards normal levels.

Pressor and depressor catecholamines similarlyaffected the transport of the large molecular tracerferritin which enters the myocardial interstitiumexclusively by plasmalemmal vesicles (Fig. 5). Upperset of pictures are electron micrographs from myo-cardium 20 min after ferritin injection. As comparedwith control (a) there was an increased number offerritin molecules in the myocardial interstitiumfollowing norephinephrine infusion (b). Whereasthe difference between the control and the norepine-phrine-treated rats for ferritin was less striking thanfor peroxidase transport, at low blood pressure levelsproduced by isoproterenol, there was a striking

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diminution of ferritin molecules in the myocardialinterstitium (c, control; d, isoproterenol, 60 mincirculation time).

Conclusions(1) Structural-functional correlative studies sug-

gest a relationship between blood pressure changes,coronary transendothelial protein transport andalteration of cardiac muscle cells following com-parable low dose intravenous infusion of pressor anddepressor catecholamines.

(2) Isoproterenol, a depressor amine, induced adiminution of transendothelial passage of finestructural protein tracers, that was more marked forperoxidase than for ferritin. This phenomenon wasreversible with return to normal blood pressure.Norepinephrine, a pressor amine, augmented thetransendothelial passage of both peroxidase andferritin.

(3) In isoproterenol model, decreased transen-dothelial transport and myocardial perfusion mayindicate the role of an additional factor in the com-plex mechanism whereby this catecholamine bringsabout myocardial hypoxia. Observation in the re-cuperation phase furnished further component in thechain of events leading to cardiac muscle cell injury.Following return of blood pressure towards normallevels, increased sarcolemmal permeability wasreflected by the intrasarcoplasmic presence ofextracellular diffusion tracers. Similar membranealteration was observed in the early stage ofnorepine-phrine experiment. These observations suggest thatearly membrane permeability change may be thekey factor in catecholamine myocardial injury.The experimental protocol presented may give

further information concerning the development andevolution of cardiac muscle cell lesions produced byother types of injury. It is possible to hypothesize, in

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Study of cardiac muscle cell injury 339

the meantime, that in humans subjected to stress,release of excessive amount of catecholamine mayalso produce functional alteration of coronary capil-laries. Subsequent change of sarcolemmal membranepermeability leads to intrasarcoplasmic depositionofproteinaceous substances. The evolution of cardiacmuscle cell injury in heart muscle following admini-stration of catecholamines was the subject of aprevious presentation at the 6th Annual Meeting ofthe International Study Group for Research inCardiac Metabolism in Freiburg. The results of thefunctional-correlative study outlined in the presentpaper suggest that coronary microcirculatory changesand membrane permeability alteration representimportant components in a common mechanismthat leads to myocardial changes which developwithout narrowing or obstruction of coronaryarteries.

ReferencesBOUTET, M., HCJTTNER, I. & RONA, G. (1973) Aspect micro-

circulatoire des 1lsions myocardiaques provoquEes par

l'infusion de catecholamines. Etude ultrastructurale al'aide de traceurs de diffusion. 1. Isoprot6r6nol. Pathologieet biologie (Paris), 8, 811.

BOUTET, M., HOTTNER, I. & RONA, G. (1974) Aspect micro-circulatoire des lesions myocardiques provoqu6es parl'infusion de cat6cholamines. Etude ultrastructurale al'aide de traceurs de diffusion. 2. Nor&pin6phrine. Patho-logie et biologie (Paris), 5, 377.

HUTTNER, I., BOUTET, M. & MORE, R.H. (1973a) Studies onprotein passage through arterial endothelium. J. Struc-tural correlates of permeability in rat arterial endothelium.Laboratory Investigation, 28, 672.

HUTTNER, I., BOUTET, M. & MORE, R.H. (1973b) Studies onprotein passage through arterial endothelium. II. Regionaldifferences in permeability to fine-structural proteintracers in arterial endothelium of normotensive rats.Laboratory Investigation, 28, 678.

HUTTNER, I., BOUTET, M., RONA, G. & MORE, R.N. (1973)Studies on protein passage through arterial endothelium.1ll. Effect of blood pressure levels on the passage of fine-structural protein tracers through rat arterial endothelium.Laboratory Investigation, 29, 536.

RONA, G. (1971) Cardiac adaptation to insult. Symposium onMetabolism and disease, Health and Welfare Canada, p. 50.Ottawa, Canada.

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