Sequential changes of energy metabolism and mitochondrial function in myocardial infarction induced...

Sequential changes of energy metabolism andmitochondrial function in myocardial infarctioninduced by isoproterenol in rats: a long-termand integrative study

Victoria Chagoya de Sánchez, Rolando Hernández-Muñoz,Fernando López-Barrera, Lucía Yañez, Susana Vidrio, Jorge Suárez,Ma. Dolores Cota-Garza, Alberto Aranda-Fraustro, and David Cruz

Abstract: Acute myocardial infarction is the second cause of mortality in most countries, therefore, it is important to know theevolution and sequence of the physiological and biochemical changes involved in this pathology. This study attempts tointegrate these changes and to correlate them in a long-term model (96 h) of isoproterenol-induced myocardial cell damage inthe rat. We achieved an infarct-like damage in the apex region of the left ventricle, occurring 12–24 h after isoproterenoladministration. The lesion was defined by histological criteria, continuous telemetric ECG recordings, and the increase inserum marker enzymes, specific for myocardial damage. A distinction is made among preinfarction, infarction, andpostinfarction. Three minutes after drug administration, there was a 60% increase in heart rate and a lowering of bloodpressure, resulting possibly in a functional ischemia. Ultrastructural changes and mitochondrial swelling were evident fromthe first hour of treatment, but functional alterations in isolated mitochondria, such as decreases in oxygen consumption,respiratory quotient, ATP synthesis, and membrane potential, were noticed only 6 h after drug administration and lasted until72 h later. Mitochondrial proteins decreased after 3 h of treatment, reaching almost a 50% diminution, which was maintainedduring the whole study. An energy imbalance, reflected by a decrease in energy charge and in the creatine phosphate/creatineratio, was observed after 30 min of treatment; however, ATP and total adenine nucleotides diminished clearly only after 3 h oftreatment. All these alterations reached a maximum at the onset of infarction and were accompanied by damage to themyocardial function, drastically decreasing left ventricular pressure and shortening the atrioventricular interval. Duringpostinfarction, a partial recovery of energy charge, creatine phosphate/creatine ratio, membrane potential, and myocardialfunction occurred, but not of mitochondrial oxygen consumption, rate of ATP synthesis, total adenine nucleotides, ormitochondrial proteins. Interesting correlations of the sequential changes in heart and mitochondrial functions with energymetabolism were obtained at different stages of the isoproterenol-induced cardiotoxicity. These correlations could be useful tostudy and understand the cellular events involved in this pathology.

Key words: cardiotoxicity, myocardial ischemia, physiopathology, heart energy balance, heart mitochondria, telemetry system.

Résumé: L’infarctus aigu du myocarde est la deuxième cause de mortalité dans la plupart des pays; par conséquent, ilapparaît important de connaître l’évolution et la séquence des variations biochimiques et physiologiques mises en cause danscette pathologie. La présente étude tente d’intégrer ces variations et de les corréler dans un modèle de longue durée (96 h) delésion cellulaire myocardique induite par l’isoprotérénol chez le rat. Nous avons obtenu une lésion de type infarctus dans larégion de l’apex du ventricule gauche, 12–24 h après l’administration de l’isoprotérénol. La lésion a été définie par descritères histologiques, des enregistrements ECG continus par télémétrie et l’augmentation d’enzymes marqueuses sériques,spécifiques à la lésion myocardique. Une distinction est faite entre préinfarctus, infarctus et postinfarctus. Trois minutes aprèsl’administration du produit, la fréquence cardiaque augmente de 60% et la tension artérielle diminue, ce qui provoqueprobablement une ischémie fonctionnelle. Des variations ultrastructurales et le gonflement des mitochondries ont étémanifestes dès la première heure de l’intervention, mais des modifications fonctionnelles dans les mitochondries isolées, tellesque des diminutions de la consommation d’oxygène, du quotient respiratoire, de la synthèse de l’ATP et du potentiel de

Received September 20, 1996.

V. Chagoya de Sánchez,1 R. Hernández-Muñoz, F. López-Barrera, L. Yañez, and S. Vidrio.Departamento de Bioenergética, Institutode Fisiología Celular, UNAM, Apartado Postal 70-243, 04510 México D.F., México.J. Suárez and Ma. D. Cota-Garza.Departamento Farmacología, Instituto Nacional de Cardiología “Ignacio Chávez,” Juan Badiano 1,Tlalpan 14080, México D.F., México.A. Aranda-Fraustro and D. Cruz. Departamento Patología, Instituto Nacional de Cardiología “Ignacio Chávez,” Juan Badiano 1,Tlalpan 14080, México D.F., México.

1 Author to whom all correspondence should be addressed at the Instituto de Fisiología Celular, Universidad Nacional Autónoma deMéxico, Apartado Postal 70-243, 04510 México D.F., México.

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membrane, ont été notées seulement 6 h après l’administration du produit et ont duré plus de 72 h. Les protéinesmitochondriales ont diminué après 3 h, atteignant une diminution de près de 50% qui s’est maintenue durant toute l’étude. Undéséquilibre énergétique, reflété par une diminution de la charge énergétique et du rapport créatine phosphate/créatine, a étéobservé après 30 min; toutefois, l’ATP et les adénonucléotides totaux n’ont vraiment diminué qu’après 3 h. Toutes cesvariations ont atteint un maximum au début de l’infarctus et ont été accompagnées d’une altération de la fonctionmyocardique diminuant significativement la pression ventriculaire gauche et raccourcissant l’intervalle atrioventriculaire. Enpostinfarctus, un rétablissement partiel de la charge énergétique, du rapport créatine phosphate/créatine, du potentiel demembrane et de la fonction myocardique a été observé, ce qui n’a pas été le cas de la consommation d’oxygènemitochondriale, du taux de synthèse de l’ATP, des adénonucléotides totaux ou des protéines mitochondriales. Des corrélationsintéressantes des variations séquentielles des fonctions mitochondriales et cardiaques avec le métabolisme énergétique ont étéétablies à différentes phases de la cardiotoxicité induite par l’isoprotérénol. Ces corrélations pourraient être utiles pour étudieret comprendre les processus cellulaires mis en cause dans cette pathologie.

Mots clés: cardiotoxicité, ischémie myocardique, physiopathologie, équilibre énergétique thermique, mitochondriecardiaque, système de télémétrie.[Traduit par la Rédaction]

Introduction

Acute myocardial infarction is a significant cause of humanmortality (Tunstall-Pedoe et al. 1994); however, over the lastyears this situation has diminished mainly as a result of im-proved technology and the availability of more effective andexpensive therapies (Wittels et al. 1990). To know the mecha-nism by which infarction occurs, as well as to outline strategiesfor its prevention or treatment, it is necessary to have an ex-perimental model easy to manipulate and reproduce. Since theearliest observations of Rona et al. (1959) establishing the factthat isoproterenol, a syntheticβ-adrenergic agonist, inducesmyocardial infarction in rats, several groups have used thisexperimental approach to study the mechanism of cardiotoxic-ity induced by catecholamines and isoproterenol (Singh et al.1988; Stanton et al. 1969) and the cardioprotection elicited bysome compounds, such as ribose (Zimmer and Ibel 1983),adenosine (Singh et al. 1988), adenine, and inosine (Zimmerand Schneider 1991). Nonetheless, the different experimentalconditions used, related to the type of animals, the isoproter-enol dose, and the administration schedule, and the lack ofintegration of morphological (Rona et al. 1959), mitochondrialfunction (Uyemura and Curti 1991), and hemodynamic studies(Vleeming et al. 1990) have made it difficult to integrate thiswhole body of valuable information. In addition, most of thesestudies have been short-term experiments, hindering the evalu-ation of the time course of this pathology. This study wasaimed at establishing a long-term, integrated model of isopro-terenol-induced myocardial cell damage in rats. The infarct-like damage was defined by histological, hemodynamic, andbiochemical criteria, distinguishing the main stages of cardio-toxicity: preinfarction, infarction, and postinfarction. An im-portant objective of this study was to establish a relationbetween the structural, biochemical, and physiological changesand the sequential modifications in energy metabolism andmitochondrial function before, during, and after the infarction,to elucidate which changes trigger the infarct-like damage andwhich persist.

Materials and methods

MaterialsEnzymes, coenzymes, isoproterenol, fatty acid free albumin, and the

kits for enzyme determinations were from Sigma Chemical Co.(St. Louis, Mo.). All other reagents were obtained from Merck(Mexico).

Animal treatmentMale Wistar rats, weighing 250–300 g and provided with food andwater ad libitum, were injected subcutaneously (s.c.) with (–)-isopro-terenol hydrochloride at a 67 mg/kg body weight dose, between 08:00and 09:00. This dose was chosen experimentally as it lowers mortalityand allows study of the experimental animals for 96 h. Control ani-mals received a s.c. administration of saline. Experimental animalswere killed at 3, 6, 12, 24, 48, 72, and 96 h after treatment, exceptthose animals chosen for evaluation of hemodynamic parameters,ECG, and temperature by radiotelemetry. After killing the animal, ablood sample was taken to obtain serum. Three samples from the heartwere obtained: (i) for histological studies, (ii ) for electron micros-copy, and (iii ) for mitochondrial preparations or perchloric acid ex-tracts. Another set of experiments, under similar conditions, wasperformed to evaluate the heart function in a Langendorff preparation(see heart perfusion experiments).

Animals were cared for in accordance with the principles of theGuide to the Care and Use of Experimental Animalspublished by theCanadian Council on Animal Care.

Histological studiesFour rats, one control and three experimental animals, were used foreach experimental time. The heart was removed immediately afterdeath and three symmetrical pairs of slices (specular image) were cuttransversely from the base, middle part, and apex. One slice was fixedin 10% neutral buffered formalin. After embedding the tissue in par-affin, sections were cut at 4µm thickness and stained with hema-toxylin–eosin and Masson’s trichrome. The other slice was used forelectron microscopy, fixed in 2.5% glutaraldehyde, and a small partof this slice was postfixed in 1% osmium tetroxide in phosphate buff-er at pH 7.4 and stained with uranyl acetate and lead citrate. Thesections were examined with a Zeiss, EM-10A, electron microscope.Sections of at least two blocks from each slice were examined. Quan-titative and qualitative histological evaluations were performed in adouble blind manner. Quantitation of damage was established with alight microscope locating and representing the lesions in a transversalcut diagram of the heart at the ventriclar level (Todd et al. 1985a).Qualitative evaluation was made with light and electron microscopy,considering the following types of damage: elongation, undulation ofthe fibers, and formation of contractile band lesions as characteristicof the preinfarction stage; coagulative necrosis and fragmentation offibers as representative of damage of infarction; and the presence ofmacrophages, fibroblasts, and collagen fibers as pertaining to a

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postinfarction stage. Electron microscopy was used to correlate sub-cellular alterations.

Hemodynamic studies, ECG, and body temperatureThese parameters were studied by radiotelemetry in freely movingconscious animals with Data Sciences International equipment(Brockway and Hassier 1993), using a TL11M2-C50-PXT implant tomonitor blood pressure, systolic, diastolic, and mean pressures, heartrate, ECG, and temperature. To implant transmitters, rats (250–300 g)were anesthetized with an i.p. injection of ketamine plus xylazine(respectively, 80 and 10 mg/kg body weight). Under aseptic condi-tions, a midline abdominal (4–5 cm) incision was made, the intestinewas retracted, and the lower abdominal aorta was isolated. A smallpuncture hole was made in the aorta at the bifurcation level. The bloodpressure catheter of the transmitter was inserted 8 mm into the vessel,the area of insertion was dried, and the catheter was sealed with amedical-grade tissue adhesive. The intestines were inserted back inplace and the body of the transmitter was sutured to the abdominalwall at the incision site. To monitor ECG signals, the electrodes wereplaced subcutaneously, one at the right shoulder and the other at theleft leg in small incisions located at the desired sites. The distal end(1 mm) of the exposed stainless-steel coil was inserted into a siliconetip cover to reduce tissue irritation. The electrode was sutured to thetissue to prevent migration. These animals were allowed to recoverfor at least 1 week. Twenty-four-hour recordings were taken as con-trols of the parameters to be studied. Afterwards, a saline injection(s.c.) was given to obtain a baseline of the recorded stress response.Isoproterenol was then s.c. administered, and recordings of the stud-ied parameters were immediately started, lasting for up to 96 h aftertreatment.

Biochemical studies

Serum levels of myocardial damage marker enzymesBlood samples were taken from the neck to determine serum enzymeactivities. Serum levels of creatine phosphokinase (CK) (EC 2.7.3.2)and its heart isoenzyme (CK-MB), as well asα-hydroxybutyratedehydrogenase (α-HBDH) (EC 1.1.1.30), were determined usingconventional diagnostic kits. Aspartate aminotransferase (AST)(EC 2.6.1.1) was measured by the method of Bergmeyer and Bernt(1965).

Parameters of mitochondrial functionThe heart mitochondrial fraction was isolated from heart homogen-ates in 180 mmol/L KCl, 10 mmol/L EDTA, and 0.5% de-fatted albu-min, at pH 7.2. The homogenate was centrifuged at 1500× g for10 min, and the supernatant was filtered through a cheesecloth andspun at 8500× g for 10 min. Mitochondria were washed three timeswith the same solution and suspended in 0.5 mL of 180 mmol/L KCl,0.5% albumin. Protein content was determined according to Lowryet al. (1951). Mitochondrial respiration and phosphorylation were re-corded polarographically with a Clark-type oxygen electrode in amedium containing 250 mmol/L sucrose, 0.5 mmol/L EDTA, and3.0 mmol/L phosphate buffer, pH 7.4; phosphorylation was initiatedwith the addition of 250µmol/L ADP. The final concentration of thedifferent substrates was 5 mmol/L. The membrane potential wasmeasured by monitoring the movements of tetraphenylphosphoniumacross the mitochondrial membrane and the pH difference across theinner mitochondrial membrane by the equilibrium distribution of ra-diolabeled acetate, as previously described (Hernández-Muñoz et al.1992; Valcarce et al. 1988). The intramitochondrial volume was esti-mated by the differential distribution of tritiated water and [14C]su-crose in isolated mitochondria, according to the Valcarce et al.modification (Valcarce et al. 1988) of Rottemberg’s method. The∆ψ and ∆pH were calculated using the Nernst equation (Nicholls1982). Mitochondrial recovery and the amount of protein per gram oftissue were calculated using the cytochrome oxidase activity as a

marker enzyme, as described before (Ma et al. 1989); activity ofcytochrome oxidase was determined by the method of Rafael (1983).During the first 6 h after isoproterenol administration, mitochondrialyield was essentially the same as in controls. Mitochondrial yieldsignificantly decreased (15± 2.1%) thereafter in hearts fromisoproterenol-treated rats, indicating a “selection” of mitochondrialpopulation.

Analytical proceduresFor adenine nucleotide determinations, approximately 300 mg sam-ples of the rat heart were immediately homogenized in 3 mL ice-cold8% perchloric acid; for creatine and phosphocreatine determinations,the heart sample was extracted with 6% perchloric acid and 10%methanol (Kapelko et al. 1988). The homogenates were centrifugedat 9000× g for 10 min at 4°C. The acid extract was divided intofractions and maintained frozen until determinations were made. Acidextract was neutralized with 4 M K2CO3. Adenine nucleotides(Hoffman and Liao 1977), creatine, and phosphocreatine (Juenglingand Kammermeier 1980) were quantified by reversed-phase high per-formance liquid chromatography. Energy charge (EC) was calculatedaccording to Atkinson (1968).

Heart perfusion experimentsThe experimental procedure has been described previously in detail(Suárez and Rubio 1991). Briefly, animals were anesthetized with ani.p. injection of ketamine plus xylazine (respectively, 80 and10 mg/kg body weight) and heparin (500 U). The heart was removedand retrogradely perfused via a nonrecirculating perfusion system atconstant flow. Perfusion medium consisted of Krebs–Henseleit solu-tion (K-H), with the following composition (mmol/L): NaCl, 117.8;KCl, 6; CaCl2, 1.6; NaHCO3, 25; NaH2PO4, 1.2; NaEDTA, 0.0027;and glucose, 5.0. This solution was equilibrated with 95% O2 – 5%CO2 at 37°C and pH of 7.4. All experiments were performed keepinga constant coronary flow of 10 mL/min. Coronary perfusion pressurewas recorded continuously via a side arm of the perfusing cannula(control value of 46.3± 3.1 mmHg; 1 mmHg= 133.3 Pa). One pair ofstimulating electrodes was placed in the apex of the right atrium, andelectric square pulses of 2.0 ms duration and two times the thresholdwere applied. To record the electrocardiogram, one electrode wasplaced in the right atrium and a second electrode in the left ventricle.These two electrodes were connected to an oscilloscope synchronizedwith the atrial pacing stimulator, whereas the atrioventricular delay(A-V delay, ms) was continuously monitored and measured as thetime interval between the application of the stimulus to the atrium andthe initiation of the rising phase of the ventricular signal. The timebetween the application of the stimulus and the atrial electrogramremained constant (18.3± 1.0 ms) throughout all the manipulations.Left ventricular pressure (LVP) was measured introducing a latexballoon into the left ventricle via the left atrium. Diastolic pressurewas adjusted to about 10 mmHg and the developed pressure continu-ously monitored.

StatisticsAll values are expressed as means± SEM. The significance of thedifference was assessed by Student’st test applied to paired compari-sons by one-way ANOVA, with a significance level ofp < 0.05.

Results

Histological studiesLight microscopy showed a sequential damage along the 96 hafter the s.c. isoproterenol injection (Table 1). Elongation andondulation of the fibers and of the contraction bands werenoted within the first hour, being more evident between 3 and6 h, and disappearing almost 12 h after the treatment. Coagu-lation necrosis and fiber fragmentation were found between 12

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and 24 h (Table 1). Inflammation and edema were also associ-ated with acute myocardial infarction. The presence of macro-phages, fibroblasts, and collagen was evident between 48 and96 h (Table 1, Fig. 1C). Ultrastructural alterations were evi-dent after the first hour of treatment. These consisted of en-largement of Z lines, mitochondrial swelling accompanied bya clearing of the matrix with cristae displacement, glycogengranule reduction, and condensation of nuclear chromatin inperipheral granules. An important damage in the sarcomere,characterized by prominent contraction bands and disruption

of the myofibrils, was observed at 3 h after drug administration(Fig. 2B). A more severe mitochondrial damage was observedafter the first 6 h of treatment. Mitochondria presented cristaerectification and occasional electron-dense deposits, which in-creased markedly at 12 h of isoproterenol administration,mainly as donut-shaped granular dense bodies in the mito-chondrial matrix (Fig. 2D).

All the experimental animals showed an infarct-like dam-age of the circumferential type in the subendocardium, at theapex region of the left ventricle. The subepicardium damage

Time of treatment (h)

Type of damage 1 3 6 12 24 48 72 96

Elongation and ondulation of fibers +++ +++ ++ + – – + +Necrosis and contraction bands – + +++ ++ + – – –Coagulative necrosis – – – + ++ – – –Fragmentation of myofibrils – – – – + ++ – –Polymorphonuclear cells – – + ++ +++ + – –Macrophages – – – – + +++ ++ +Fibroblasts and collagen fibers – – – – – + ++ +++Edema – – + ++ +++ ++ + –

Note: Animals received an injection (s.c.) of isoproterenol and were killed at different times after drug administration; total damage after s.c. administration ofisoproterenol was taken as 100%;+, 25%;++, 50%;+++, 75%; –, not observed.

Table 1.Histologic evaluation of the myocardial lesion induced by isoproterenol treatment.

Fig. 1.Histological findings after isoproterenol administration. (A) Individual coagulation necrosis, 6 h after isoproterenol administration. Lossof cross striation and cytoplasmic acidophilia. Hematoxylin–eosin,×40. (B) Focal coagulation necrosis, 24 h after isoproterenol administration,neutrophil infiltration is prominent. Hematoxylin–eosin,×16. (C) Mononuclear infiltration at 48 h after drug administration, there aremacrophages, fibroblasts (*), and myocardial cell fragmentation. Hematoxylin–eosin,×40. (D) Subendocardial fibrosis, 96 h after drugadministration, light areas. Masson trichrome technique, 10×.


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was minimal, and there was no transmural infarction, since thelesion affected, on average, one-third of the thickness of theventricular wall. None of the control animals presented evi-dence of myocardial damage.

According to the histological observations, we consideredcoagulative necrosis as an indicator of infarction, occurringalways between 12 and 24 h after isoproterenol administra-tion. The alterations observed before this time could be con-sidered as representing a preinfarct stage (0–12 h). Thepostinfarct stage was evidenced by the onset of cicatrization(48–96 h).

Blood pressure and heart rateThese parameters were monitored continuously by telemetry.The animal received a subcutaneous saline injection 24 h be-fore the treatment; discrete stress-induced changes were ob-served in blood pressure and heart rate (data not shown).Subcutaneous administration of isoproterenol resulted in astrong and immediate increase (60%) in heart rate during thefirst 5 min (Fig. 3A). The high heart rate was maintained for10 min, followed by a slight but constant decrease (10–15%)during 36 h, reaching the control level within 72 h of the treat-ment (Fig. 3B). Variations in blood pressure, systolic and dia-stolic, followed a specular image of the heart rate changes.Figure 3 shows the mean systolic blood pressure and heart rate

values of five experimental observations. The immediate in-crease in heart rate and the lowering of blood pressure mighthave resulted in myocardial ischemia sustained at least for 2 h.

Electrocardiographic changesThe telemetry system also registered continuously the ECGchanges (Fig. 4). Sinus tachycardia was observed from the first10 min up to 72 h of treatment. Advanced right bundle branchblock, characterized by a broad slurred S wave in the left ven-tricular morphology, appeared concomitantly with tachycardiaduring the first 5 h. Atrioventricular block was established af-ter 90 min of the treatment, lasting for 20 h. Data of subendo-cardial infarction appeared 12 h post-injection, characterizedby a sudden ST segment elevation and a broad and deepQ wave with transmural injury. The ST segment elevation re-turned to the baseline at 48 h post-treatment, but the Q wavealterations persisted, and a deep, symmetrical, and negativeT wave transmural ischemia appeared. ECG tracings were nor-mal at 96–120 h post isoproterenol treatment.

Biochemical studies

Enzyme releaseThe results of the effect of isoproterenol on the serum markerenzymes of myocardial damage (CK, CK-MB,α-HBDH, and

Fig. 2.Ultrastructural findings after isoproterenol administration. (A) Portion of myocardial fiber from a control animal. Electron microscopy(1000×). (B) Three hours after isoproterenol administration. Note the formation of contraction bands (*). 3000×. (C) At 6 h after isoproterenol,note the disruption of myofibrils and polymorphonuclear cells in the interstitial space. 3000×. (D) Mitochondria after 12 h of isoproterenol,donut-shaped (arrow) granular dense bodies develop in the matrix. These densities have intrinsic electron opacity and are more opaque than theamorphous densities, which are also present (arrowhead). 6000×.


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AST) reflected a similar profile for CK and CK-MB, reachingthe maximum after 3 h of the treatment (control 110± 9.2 vs.220± 23.0, n = 5), corresponding to the preinfarction stage;α-HBDH and AST were slowly released and their maximalserum activities were found after 12 h of treatment (control22 ± 1.9 vs. 50± 8.0 and 110± 7.0 vs. 260± 16.8, respectively,n = 5), corresponding to the infarction time.

Mitochondrial functionAlthough ultrastructural changes and mitochondrial swellingwere evident from the first hour of isoproterenol treatment,measurement of the ADP-stimulated oxygen consumption(state 3), in isolated mitochondria from treated animals, eitherwith malate–glutamate (Table 2) or succinate (not shown) assubstrate, revealed a significant decrease in oxygen consump-tion starting at 6 h after treatment (Table 2) and correlated withconsiderable mitochondrial damage. A progressive diminutionof this parameter continued for up to 72 h after treatment;thereafter, oxygen consumption recovered with both sub-strates. No significant changes were observed in nonstimulatedoxygen consumption (state 4, not shown). Hence, the de-creased values in the respiratory quotient were mainly due tothe diminished state 3 in these animals (Table 2). The mito-

chondrial ADP/O ratio for both substrates, glutamate–malate(G-M) or succinate, presented a significant diminution only at12 h but not at other times (control 2.77± 0.2 vs. 2.02± 0.19for G-M) of treatment, suggesting that mitochondrial couplingis moderately affected during the onset of myocardial in-farction. However, the calculated rate of ATP synthesis withboth substrates was significantly diminished, starting between6 and 72 h after treatment (Table 2), indicating a deficient ATPproduction by mitochondria isolated from isoproterenol-treated rats. Other parameters of mitochondrial function andthe mitochondrial amount per gram of tissue in hearts fromisoproterenol-treated rats are also given. The intramitochon-drial volume was nearly constant, except for a significant dimi-nution at 24 and 48 h of treatment (not shown). At the time atwhich myocardial infarction occurred (12 h), the electro-chemical potential across the membrane (∆ψ) diminished sig-nificantly, but was promptly restored (Table 2). However,during the recovery period (postinfarction), this parameter wasagain diminished, remaining low until the end. The same pro-file was shown for the pH gradient across the mitochondria(∆pH), which was significantly decreased at later times (notshown). Variations in heart mitochondria proteins were moredrastic during the onset of myocardial infarction. A progres-sive loss of mitochondria was also observed starting at 6 h,

Fig. 3. In vivo changes of heart rate and blood pressure induced byisoproterenol. Rats received an injection (s.c.) of isoproterenol attime 0, as described in Materials and methods. The response wasmonitored continuously by a telemetry system. (A) First 10 minevolution of a typical experiment. (B) Time course of the responseup to 96 h after drug administration. Data points are means± SEM(n = 5).

Fig. 4.Sequence of ECG changes during isoproterenol-inducedinfarction. ECG was monitored by the telemetric system asdescribed under Materials and methods. These results correspond toa representative experiment. Times after s.c. injection ofisoproterenol are indicated.


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which did not revert during the period of myocardial recovery.These findings support the decreased mitochondrial oxidativefunction found in our experimental groups.

Energy metabolismEvaluation of the energy balance in vivo during the evolutionof the isoproterenol-induced cardiotoxicity revealed impairedmitochondrial structure and function in the experimental ani-mals. Table 3 gives modifications of adenine nucleotides andenergy parameters in the hearts from animals treated with iso-proterenol. Total adenine nucleotides decreased significantlystarting 3 h after treatment, reaching a minimum at 24 h, afterwhich a recovery was observed; however, only 56% of totaladenine nucleotides was found after 96 h of drug administra-tion. This diminution resulted from a marked decrease in ATP,moderate diminution of ADP (6–96 h), and increased AMPobserved at most of the times tested. These changes are re-flected in the energy parameters. During the first 24 h, wheninfarction occurs, the changes in adenine nucleotides were ac-companied by an significant decrease in EC, from 0.80 to 0.56.During the recovery time, total adenine nucleotides did notreach normal values; however, the relation among ATP, ADP,and AMP resulted in an amelioration and further normalizationof the energy charge. During the first 6 h, a 50% elevation ofinorganic phosphate (Pi) occurred (not shown) (control value12.7± 1.6,n = 8), possibly reflecting adenine nucleotide deg-radation. At the other times, Pi values remained almost con-stant. Values of other energy parameters, such as ATP/ADP

ratio and the phosphorylation potential (ATP/ADP⋅Pi) fol-lowed a pattern similar to the energy charge (not shown). It isworth mentioning that at early times (30 and 60 min after iso-proterenol administration), the decrease in energy charge wasevident (0.60), although total adenine nucleotides did notchange markedly. The diminution in adenine nucleotides andenergy parameters in the heart of isoproterenol-treated rats wasaccompanied by a marked decrease in blood ATP and in-creased levels of ADP and AMP, with a diminution of totaladenine nucleotides and of energy charge at 3 to 24 h of treat-ment, followed by a recovery to normal values at 48 h (notshown); the latter suggests a general hypoxic state during thepreinfarction and infarction stages.

Modifications of heart creatine (Cr) and phosphocreatine(CrP), during the onset of the experimental myocardial in-farction, were also studied (Table 4). A significant decrease inCr + CrP was found at 24 and 48 h of the treatment, but theCrP/Cr ratio did not change significantly, whereas a markeddecrease in CrP/Cr ratio (0.23) was observed within 30 and60 min of the treatment (not shown), reaching a minimum af-ter 3 h (Table 4). During the recovery time (48–96 h) the levelof Cr + CrP as well as the CrP/Cr ratio were normal, differingfrom the adenine nucleotide system, which did not recover thenormal values of total adenine nucleotides.

Functional studiesMyocardial function was evaluated by measuring left ventricu-lar pressure and the A-V interval in isolated perfused hearts

Time aftertreatment (h)

State 3(natoms O2⋅min–1⋅mg–1) RC

ATP synthesis(nmol⋅min–1⋅mg–1) ∆Ψ

Mitochondrialcontent

0 115.0±6.7 9.7±0.05 319±21 163±7 50.7±3.93 120.2±14.5 9.0±0.90 305±43 169±4 45.1±6.56 91.9±11.1 6.2±0.60* 243±43 180±3 31.4±3.1*

12 88.9±11.3 6.8±0.80* 180±20* 121±10* 24.3±3.5*24 78.8±4.8* 8.3±0.40* 239±19* 157±8 21.1±2.9*48 60.6±5.4* 6.3±0.60* 148±15* 163±2 21.0±1.6*72 36.8±4.8* 5.9±0.50* 112±14* 142±2* 22.8±2.1*96 69.1±2.0* 5.1±0.40* 192±15* 120±3* 24.0±2.0*

Note: Values are means± SEM from at least six independent observations per time. Glutamate–malate was used as substrate. ATP synthesis was calculatedfrom the product of the rate of ADP-stimulated oxygen consumption by the ADP/O ratio. Time 0 corresponds to the values obtained from control animals(saline).Ψ is the electrochemical potential across mitochondrial membrane (negative inside). The content of mitochondrial protein (mg/g) per heart wascalculated using the cytochrome oxidase activity as specific marker, as described under Materials and methods. RC, respiratory control state 3/state 4.

*Significant statistical difference against the control group (p < 0.01).

Table 2.Oxygen consumption, phosphorylating capacity, and electrochemical potentials of isolated mitochondria and mitochondrial content inrat heart from isoproterenol-treated rats.

Time aftertreatment (h) ATP

ADP(µmol⋅g–1) AMP AN EC

0 5.9±0.50 2.2±0.2 0.6±0.10 8.7±0.5 0.80±0.023 2.5±0.20* 2.7±0.4 1.2±0.20* 6.4±0.7 0.60±0.026 2.3±0.20* 1.7±0.05* 1.2±0.20* 5.2±0.2* 0.60±0.03

12 2.2±0.20* 1.5±0.20* 1.2±0.20 4.9±0.5* 0.60±0.0624 1.2±0.10* 0.8±0.06* 0.8±0.10* 2.9±0.2* 0.56±0.0248 2.9±0.10* 1.2±0.05* 0.4±0.06 4.5±0.1* 0.77±0.0172 2.7±0.06* 1.6±0.09* 0.9±0.08* 5.2±0.1* 0.67±0.0196 2.9±0.90* 1.3±0.08* 0.4±0.06 4.6±0.4* 0.77±0.01

Note: AN, sum of adenine nucleotides; EC, energy charge (ATP+ ½ADP/ AN). Values are means± SEM,n = 4.*p < 0.05.

Table 3.Changes in rat heart adenine nucleotides during the experimental infarction induced by isoproterenol.


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from animals treated at different times with isoproterenol(Fig. 5). During the preinfarction and infarction stages, theLVP dropped drastically during the first 6 h of treatment, re-maining depressed for up to 48 h, without reaching the controlvalues at the last time tested (96 h). This altered myocardialfunction correlated with a shorter A-V interval observed dur-ing the first 6 h after injection, reaching a minimum value at48 h, and remaining low even during the recovery period.

Discussion

The infarct-like lesion induced by isoproterenol in rats, origi-nally described by Rona et al. (1959) and extensively studiedby his group for more than 25 years (Rona 1985), has providedimportant information on this model; however, a long-termintegrated study is still missing. Only, integrated short-termstudies of the isoproterenol-induced cardiotoxicity were per-formed by Rona (1985), correlating myocardial lesions withelectrocardiographic changes in the dog; Todd et al. (1985b)also correlated the contraction band lesions induced by isopro-terenol with the ECG, as well as with hemodynamic and somebiochemical changes, in dogs, exposed to continuous infusionof isoproterenol. The present work is the first one to offer along-term study (0–96 h), integrating histological, physiologi-cal, and biochemical aspects of an experimental model of myo-cardial cell damage induced by isoproterenol in rats under thesame experimental conditions. This model has the advantagesof easy manipulation, reproducibility, and low mortality. Theuse of a telemetric system has been very useful by offering acontinuous recording during the 96 h of ECG tracings, heartrate, blood pressure, and temperature. The results obtained of-fer a reference frame to correlate sequential physiological andmetabolic events occurring during the three defined stages ofisoproterenol cardiotoxicity: preinfarction, infarction, andpostinfarction (Fig. 6).

Experimental myocardial infarction in rats has been in-duced (Singh et al. 1988) after a daily dose of isoproterenolsulphate (85 mg/kg body weight, s.c) for 4 days. In our expe-rience, by histological and biochemical criteria, we found nofurther damage after one dose of isoproterenol hydrochloride(67 mg/kg body weight, s.c.), suggesting a desensitization ofβ-adrenergic receptors during subsequent administrations ofthe drug (Tse et al. 1979). Consequently, we decided to use adose of isoproterenol capable of inducing the three main stagesof cardiotoxicity that causes low mortality at the studied times,

and which would allow us to characterize and correlate struc-tural, metabolic, and functional changes according to the stageof cardiotoxicity.

PreinfarctionDuring the preinfarction stage (0–12 h), the first functionalevent observed after 2 min of isoproterenol administration isthe functional ischemia resulting from the immediate and sus-tained increase in heart rate, and the marked drop of bloodpressure (Fig. 3A). This feature was also evident by thechanges in the electrocardiographic tracings, showing a broadS wave, characteristic of subendocardic ischemia (Fig. 4), andby a decrease in energy parameters (Tables 3, 4). The atrioven-tricular block reflected in the ECG parameters after 1 h oftreatment is also manifested in the decrease of A-V interval inisolated perfused hearts from the animals treated with isopro-terenol (Fig. 5). These alterations in cardiac function are con-comitant with alterations in the cardiac structure (Figs. 1 and2; Table 1), evidenced by elongation and undulation of themyocardial fibers and formation of contraction bands. Sub-sequently, necrosis is evident by the loss of myocardial fibersor myocytolysis, and some ultrastructural changes, such asswelling of mitochondria, matrix clearing with displacementof cristae, and possibly a loss of mitochondrial protein(about 40%) (Table 2). The biochemical and metabolic sub-strates underlying these changes in cardiac structure and func-tion are the alterations in energy mechanisms needed tomaintain the former in physiological conditions. The increasein energy demands due to the rise of heart rate, to increase themechanical work of the contractile apparatus, and the decreasein blood flow induce an energy imbalance. This is reflected atearly times (30 min, 1 h) by a decrease in energy charge but notin ATP and total adenine nucleotides, although at later times,

Time aftertreatment (h) Creatine

Phosphocreatine(µmol⋅g–1) o CrP/Cr

0 8.5±1.00 3.7±0.90 12.77 0.443 14.0±2.80 2.3±0.20 16.40 0.166 10.6±4.17 2.6±0.60 13.20 0.2512 5.2±0.90 2.8±0.70 8.00 0.5324 4.6±0.50* 1.5±0.30* 6.10 0.3248 7.2±0.70* 3.9±0.40 11.10 0.5472 11.7±1.30 5.2±1.00 16.30 0.4696 11.0±1.30 5.4±0.90 16.40 0.49

Note: Values are means± SEM,n = 4.*p < 0.05.

Table 4.Creatine and phosphocreatine in rat heart tissue during theonset of experimental myocardial infarction with isoproterenol.

Fig. 5.Effect of isoproterenol in cardiac function tested in aLangendorff preparation. Groups of rats received an injection (s.c.)of isoproterenol and were killed at time 0 (no injection) and 12, 24,48, and 96 h after treatment. Atrioventricular (A-V) interval andleft ventricular pressure (LVP) were measured as described inMaterials and methods. Each point represents mean± SEM of theresponse from six different hearts.


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during this preinfarction stage, a simultaneous decrease in thelast two parameters was also observed (Table 3). It is interest-ing to observe that in spite of the energy imbalance detected at

30 min of isoproterenol administration, oxygen consumption,ATP synthesis, and mitochondrial proteins were decreasedclearly in isolated mitochondria only after 6 h of treatment.

Fig. 6.Temporal changes of some parameters of energy balance, mitochondrial function, and heart function along 96 h of isoproterenoladministration. Values are expressed considering the control ones as 100% and were taken as follows: panels A and B, from Table 3; panels C,D, and G, from Table 2; and panels E and F, from Fig. 5.


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This functional mictochondrial failure might impair the energyimbalance. Related with the energy balance in this stage, anincrement of creatine accompanied by a decrease of the CrP/Crratio also occurred (Table 4), which could be possibly relatedwith the loss of creatine phosphokinase from the myocardialmitochondria, considered a marker enzyme of the preinfarctionstage in this model.

InfarctionAccording to Baroldi (1975), infarction (12–24 h) was clearlydetermined histologically by the coagulative necrosis resultingfrom a severe and persisting ischemia, which produces cellulardeath with subsequent polymorphonuclear leukocyte infiltra-tion (PMN) (Figs. 1 and 2; Table 1). In our experimental ani-mals an infarct-like damage of circumferential type in thesubendocardium was observed at the apex of the left ventricle.Subendocardial infarction was also revealed in the ECG re-cording by a sudden ST segment elevation and a broad anddeep Q wave (Fig. 4). The most representative serum enzymesof acute myocardial infarction are the more slowly releasedenzymes,α-HBDH and AST, which also confirmed the pres-ence of infarction at 12 h. Although the events leading to myo-cardial infarction are not yet fully elucidated, the excessiveinotropic and chronotropic stimulation of the cardiac muscle(Fig. 3) suggests that the effect of theβ-adrenergic agonisthere studied is mediated by stimulation of the adrenergic re-ceptor, which also might be responsible for the functional hy-poxia and the alterations in energy balance. The heart rateslowly recovered its normal value and blood pressure reachedalmost normal levels, whereas the A-V interval and the LVPremained at the lowest values. The ATP value, total adeninenucleotides, energy charge, mitochondrial proteins, and therate of ATP synthesis also reached the lowest observed value.It is interesting to note that in this stage there is a significantdecrease in the mitochondrial membrane potential and that theCrP/Cr ratio reaches the normal value. Possibly, the severedamage to mitochondrial function and structure played an im-portant role in the onset of myocardial infarction, affecting theavailability of the energy required to maintain the heart func-tioning. This crucial factor could be overcome since theCrP/Cr ratio was normalized. Evidence of the importance ofthe phosphocreatine shuttle in the transfer of energy in theheart energy balance has been reported (Kammermeier 1987;Kammermeier et al. 1982).

The decrease in adenine nucleotides and energy parametersin the heart was accompanied by a marked decrease in bloodATP during the preinfarction and infarction stages, suggestingthat there is a general hypoxic condition and that these bloodparamaters could be markers of a myocardial infarction stage.

PostinfarctionOne advantage of this model is the possibility of following thephysiological recovery of the heart tissue (48–96 h). The heal-ing process is evident by the presence of macrophages, fi-broblasts, and collagen fibers (Table 1); the heart rate and theblood pressure (Fig. 3) tend to reach normal values, with aconsequent enhancement in blood flow. The LVP and the A-Vconduction in Langendorf preparations (Fig. 5) continued theirnormalization. ECG tracings showed a return to the baseline ofthe ST segment, being normal at 96–120 h (Fig. 4). The energyparameters, such as energy charge and CrP/Cr, reached almost

normal values (Tables 3 and 4). However, the mitochondrialparamaters, i.e., oxygen consumption, ATP synthesis, mem-brane potential, and mitochondrial protein content (Table 2),were not recovered. Dolgov (1975) described a diminution inthe activity of the respiratory electron chain during sustainedischemia, due to a gradual loss of intramitochondrial potas-sium. Regitz et al. (1984) and Uyemura and Curti (1991)showed changes in mitochondrial structure and function dur-ing the progression of myocardial ischemia, suggesting thatthese parameters play a key role in cellular viability duringrecovery from ischemia. Therefore, it is possible that the over-all changes observed after the myocardial infarction could berelated to a progressive physiological reperfusion mainly dueto the depressed mitochondrial function (Karmazyn 1991).

General commentsA selective loss of fast S mitochondria has been reported to beprevalent in the subendocardium of the damaged canine heartafter 1 or 2 h of myocardial infarction (Whitty et al. 1976). Inthe model here described, the loss of mitochondrial proteinscould correspond to the fast S population and was observedafter 3 h of isoproterenol treatment (11%), reaching a maxi-mum loss (60%) during the postinfarction stage (Table 2;Fig. 6), with no recovery at 96 h. This finding could suggestthe existence of a selective mitochondrial population, sincedespite the 60% mitochondrial loss, there is a recovery of theenergy balance, energy charge, and CrP/Cr ratio (Tables 3and 4) during infarction. Moreover, the decreased mitochon-drial yield found in preparations from isoproterenol-treatedrats also would stress the relevance of mitochondrial dysfunc-tion reported here, since most of the tested mitochondria wereobtained from noninjured or slightly injured cardiac tissue.

Most data obtained in this work confirm the findings ofother isoproterenol cardiotoxicity studies, but the integrationof histological, physiological, and biochemical events, as wellas the use of a telemetric system to evaluate some hemody-namic parameters, are important tools to gain new insights intothis process. Figure 6 shows the sequential changes of someof the parameters and the correlation coefficients between en-ergy parameters and heart and mitochondrial functions. Onlycorrelations with anr value above 0.6 are given (Table 5).Significant correlations were obtained between changes inATP synthesis and A-V conduction (0.91). Changes in EC,ATP/ADP ratio, and total adenine nucleotides kept a

ParameterCorrelationcoefficient p

Energy charge vs. ATP/ADP 0.918 <0.001Energy charge vs. LVP 0.830 <0.005Total nucleotides vs. LVP 0.859 <0.001Total nucleotides vs. A-V conduction 0.624 <0.01Total nucleotides vs. mitochondrial protein 0.898 <0.001ATP/ADP vs. LVP 0.873 <0.001ATP synthesis vs. A-V conduction 0.914 <0.001ATP synthesis vs. mitochondrial protein 0.850 <0.001LVP vs. A-V conduction 0.614 <0.01LVP vs. mitochondrial protein 0.768 <0.005A-V conduction vs. mitochondrial protein 0.776 <0.005

Table 5.Correlation coefficients between heart and mitochondrialfunctions and energy parameters.


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correlation of about 0.85 with the LVP. Similar correlationswere obtained between ATP synthesis and mitochondrial pro-teins, as well as for left ventricular pressure versus A-V con-duction and versus mitochondrial protein.

From these studies it is difficult to support any of themechanisms proposed for isoproterenol cardiotoxicity. Wethink that the initial functional hypoxia in the myocardium(Figs. 3 and 4; Table 4) induced by isoproterenol administra-tion could trigger alterations in energy balance by the Ca2+

overload, K+ efflux, the damage promoted by free radicalsgenerated from the oxidized products of catecholamines(Singal et al. 1982), or mitochondrial dysfunction. Furtherstudies are required to clarify these points to elucidate the cel-lular and molecular events of isoproterenol cardiotoxicity.

These results showed the critical role of mitochondrialfunction in the energy imbalance at the onset of isoproterenol-induced myocardial infarction-like lesion, i.e., the changespreceding the infarction, those concerning the production ofthe damage, and the modifications occurring post-infarction.Improvements in energy balance, EC, CrP/Cr, and ATP/ADP(Tables 3 and 5; Fig. 6) are critical for heart recovery in spiteof the fact that mitochondrial proteins, rate of ATP synthesis,and total adenine nucleotides did not reach normal values.

Acknowledgements

This study was partially supported by grants from DirecciónGeneral de Asuntos del Personal Académico (DGAPAIN-206589) and Consejo Nacional de Ciencia y Tecnología(M9109-0710). The authors acknowledge critical commentsfrom Dr. Mauricio Díaz-Muñoz and Dr. Alfredo de Michelifor the interpretation of the ECG studies, as well as the techni-cal assistance of Mrs. María Elena Miranda and Miss AngelicaRodríguez, the secretarial assistance of Mrs. Ma. ElenaGutiérrez, and the editorial assistance of Ms. Ingrid Mascher.

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