Cardiovascular Research, 1976, 10, 613-622.
Effect of chagasic sera on the rat isolated atrial preparation: immunological, morphological and functional aspects1 LEONOR S T E R I N - B O R D A , P A T R I C I O M. COSSIO, MARTHA F . G I M E N O , ALVARO L . GIMENO, CARLOS D I E Z , R U B E N P . L A G U E N S , P A T R I C I A C A B E Z A MECKERT, and R O B E R T O M . A R A N A
From the Centro de Estudios Farmacoldgicos y de Principios Naturales, and Instituto de Neurobiologia (Consejo Nacional de Investigaciones CientSficas y Tkcnicas de la Repfiblica Argentina - CONICET -), Buenos Aires, the Laboratory of Rheumatology and Immunology and the Cardiology Unit, CEMIC, Buenos Aires, and the Chtedra de Patologia II, Facultad de Medicina de La Plata, La Plata. Argentina
This work was supported in part by grants from Fundacibn Cossio, ADAR, Academia Nacional de Medicina de Buenos Aires, and CONICET (Grants 6638 and 5863/a). L.S.B.and P.C.M. are Advanced Research Fellows and M. F. G . , A.L.G.,and R.M.A. are Senior Investigators of the CONICET. Reprint requests to R.M.A., CEMIC, Sanchez de Bustamante 2560, Buenos Aires, Argentina.
The presence of an antibody reacting with and striated muscle (EVI antibody) has been described in Chagas’ disease (cOssiO et 974a). In a more recent report, it was shown that the EVI antibody reacts with antigenic determinants located in
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A U T H O R S ’ S Y N O P S I S An antibody reacting with the plasma membrane of working myocardial cells, skeletal muscle fibres, and endothelial cells (EVI antibody) has been described in the sera of patients with Chagas’ disease. In the present study of rat isolated atrial preparations beating in different media, direct immunofluorescence and ultrastructural immunohistochemical procedures indicate that the antibody can interact with the living tissue, becoming fixed to the plasma membranes. Transmission electronmicroscopy studies also showed the presence of sarcolemmal alterations. These observations suggest a possible pathogenic effect of the EVI antibody. The presence of EVI-positive sera in the beating medium leads to a significant increase in the frequency of contractions ; no significant effects of EVI-positive sera in contractile force were seen. The increase in frequency could be prevented by previous treatment with a P-adrenergic blocking agent (MJ-1999), but not by an a-blocker (phentolamine) or by an anti-histamine compound (cyproheptadine). The changes described were observed only in those atrial preparations which were beating in media containing EVI-positive sera. In those atria beating in control media (KR, KR plus normal human serum, KR plus EVI-negative chagasic serum), neither immunological nor morphological or functional changes were seen. In addition, some observations suggest that the presence of EVIpositive chagasic serum diminished atrial stimulation after added norepinephrine. These results suggest the possibility that the EVI antibody may act as a P-adrenergic agonist at the cell plasma membrane level. Such an effect might account for some of the clinical features of chronic Chagas’ heart disease.
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(KRB) (Gimeno et al, 1963) kept at room temperathe plasma membrane of working myocardial ture. The substrate for this medium was glucose at cells, skeletal muscle fibres, and vascular endo- 5.5 mmol/litre. It was gassed with a mixture of 95"; O2and 5% CO,. The atria were then separated from thelial cells (Cossio et a/, 1974b). Although it is the ventricles, carefully dissected, attached to a reasonable to assume that the plasma membranes glass holder, and immersed in a tissue chamber of different cells are, in general, able to interact filled with different nutrient fluids: ( I ) 20ml of in vivo with circulating antibodies, it has been KRB solution; (2) 17 ml of KRB plus 3 ml of fresh observed in some experimental models that normal human serum (NHS); (3) 17ml of KRB heart muscle cells did not easily react in vivo plus 3 ml of fresh chagasic EVI(-) human serum with specific antibodies (Kaplan, 1969). Further- (EVI(-)S); (4) 17 ml of KRB plus 3 ml of fresh more, a recent report showed that antibodies chagasic EVI( +) human serum (EVI( +)S). The against the Na+, K+-activated ATPase inhibit tissue bath solutions were gassed with a mixture of the enzyme activity in extracted organs, but not 95% 0, and 5% C O , throughout the experiments. Temperature and pH were 30" and 7.4. In each in the intact cell, suggesting that the antigenic bath solution 0.1 ml of Antifoam C (Sigma Chemical determinant(s) of this enzyme may be masked at Co) was added. This agent did not modify any of the the surface of the intact cells (Smith et a/, 1973). measured parameters of the beating atria. A resting The present study was performed (1) to in- tension of 750 mg was applied to the atria by means vestigate the ability of the EVI antibody to inter- of a micrometric device and their developed tension act with living myocardial cells and (2) if this and frequency were recorded by means of a force was so, to observe if this reaction could alter displacement transducer' coupled to an ink writing their physiological behaviour or modify the well- oscillograph'. Under the above procedure the isoknown actions of some hormones and drugs. The lated rat atria contracted in almost nearly isometric isolated atrial preparation was selected as the conditions. The peak tension developed by atria, over the experimental model because experimental card- resting applied tension, and their rate of contraciac anaphylaxis has been extensively studied in tion were recorded by taking tracings of the isothis model (Feigen and Prager, 1969) and also metric developed tension (IDT) and of the frebecause the atrium and the region which includes quency of contractions (FC), as previously reported the sinoatrial node is the part of the heart most (Lacuara et al, 1971). The mean absolute magnitudes of IDT and FC observed at 10 min following responsive to aoaphylaxis (Vurek et a/, 1967). isolation were taken as the initial control values (0 time). The IDT was determined in mg and the FC Material and methods measured by counting the number of atrial beats Sera per min. The preparations were then observed for Sera were obtained from 15 EVI( +) and six EVI(-) functional variations with respect to time, selecting chagasic individuals and from 10 blood donors. for recordings changes at 60, 120, and 180 min. In The EVI positivity or negativity of sera was deter- some cases the modifications observed were exmined by means of the indirect immunofluorescence pressed as the percentage of change from the level technique (Cossio et al, 1974a). All positive sera had of initial control values. However, in as much as an EVI titre 21/80, Sera were immediately used, or the first 60 min of activity might have some degree kept frozen at -7O"C, for periods up to 7 d. Comple- of spontaneous lack of stability, arising from the ment haemolytic activity of sera (CH50) was deter- trauma imposed by the dissection and the mounting mined in each sample by the method of Kabat and procedures, the absolute magnitudes of IDT and Mayer (1964) and all contained >35 CH50 units/ml FC recorded at 60 min were taken as a second (normal values 25-50 CH50 unitslml). The subjects control value and used to make the percentage and had not received any drug or therapeutic agent for absolute comparisons with records obtained at 120 the previous month. and 180 minutes. The influence on IDT and FC, elicited by noreIsolated atrial preparations pinephrine (Sigma Chemical Co), MJ-1999 (SotaMale albino rats of the Wistar strain weighing lol@, Mead Johnson), phentolamine (RegitineQ, between 200-250g were used. The animals were Ciba), or cyproheptadine (PeriactinQ, Merck, sacrificed by decapitation and the entire heart was Sharp and Dohme) was also studied over atria quickly excised and placed in a Petri dish filled with Statham, UC3-Gold Cell. a modified Krebs-Ringer-bicarbonate solution * Beckrnan, Type S-11 Dynograph.
615 Sterin-Borda, Cossio, M . Gimeno, A . Gimeno, Diez, Laguens, Meckert, and Arana overnight in 0.1 mol/litre phosphate buffer pH 7.3 and sectioned at 1 0 0 ~with a freezing microtome. The sections were treated (at 4°C) in one of the following ways: ( I ) incubation for 3 d with goat gamma globulin anti-human globulin, labelled with peroxidase; (2) incubation with rabbit gamma globulin anti-rat IgG (absorbed with normal human serum), labelled with peroxidase (1.2 mg/ml in PBS); (3) as ( I ) but previously blocked with unlabelled goat gamma globulin anti-human immunoglobulins. The material was processed as previously described (Cossio et a / , 1974b) and examined with a Philips transmission electron-microscope operated at 60 kV. The same batch of goat anti-human gamma globulin anti-serum employed for the immunofluorescence studies was used; the globulin fraction of the antiserum was obtained and IabelJed with horseradish peroxidase as reported elsewhere (Cossio et al. 1974b). Ultrastructirral microscopy Glutaraldehyde-fixed material was conventionally processed and examined by transmission electronmicroscopy. In the tissue sections morphometric study of the atrial granules was performed as previously described (Laguens, 1971).
Tissue studies Immediately after the experiments, the atria were immersed in 150ml of phosphate buffered saline (PBS) at 22°C. The atria were allowed to beat for I to 2 min after which they were washed several times for 5 min with PBS. The material was then divided into three pieces; one was immediately frozen at -2O"C, the second was fixed for 4 h at 4°C in 40/, formaldehyde in phosphate buffer 0.1 mol/litre, pH 7.3, and the third was fixed in 4q4, glutaraldehyde in phosphate buffer.
Direct immunofluorescence Cryotstat sections, 2 p thick, were cut from the frozen tissue and stained with fluorescein-labelled goat gamma globulins anti-human immuniglobulins (Cossio et al, 1974a), and anti-human C3 (Hyland Travenol Lab, Los Angeles, California, USA), as previously described (Cossio et al, 1974a).The tissues were examined with a Carl Zeiss microscope (Federal Republic of Germany) fitted with epicondenser fluorescence equipment and a phase contrast condenser.
Ultrastructural immunohistochemical procedure The atrial pieces fixed in formaldehyde were washed
Results Functional characteristics of isolated rat atrial preparations suspended in KRB, in NHS, and in EVI(-) or EVI( +) chagasic sera Table I shows that the absolute initial values (0 time) of IDT or FC of atria suspended in different media have similar magnitudes. At TABLE 1
fnitial values (means i SEM) of isometric developed tension (IDT) and frequency of contraction (FC) of isolated rat atria immersed in different suspending media* ID T
FC
__
(mgl
(beafslmin)
KRB
390 139.5 (n -25) 301 135. I (n=24) 378 1 5 3 . 0 (n=- 1 I ) 326 126.3 (n: 25)
I70 1 4 . 6 (n = 25) 175 17.1 (n = 24) 162 1 9 . 6 (n= 11) 175 +5.2 (n = 25)
Suspending media
KRB plus NHS KRB PIUS EVI (-) chagasic sera KRB plus EVl ( +) chagasic sera
*Absolute initial values recorded at LO min following isolation (0 time, see methods).
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beating in the different suspension media. Their final concentration in the tissue bath solution were: 3 . 9 lo-*, ~ 0.1, 1 . 6 ~ and 7 x 10--5mmol/ml, respectively. Freshly prepared solutions of these drugs were added to the suspending medium in a volume not higher than 0.2 ml. The addition of all these agents, except norepinephrine, was always performed at 60rnin of activity. For the experiments testing the effects of norepinephrine, a second group of atria was used. They were allowed to beat during 180min in the different media and the absolute magnitudes of IDT and FC were then recorded. Immediately a submaximal concentration of the drug was added to the bath solution, its effects registered during 10 min, and the maximal response (occurring most often at 2 or 3 min) measured, and expressed as a percentage change against the 180 min values. Results of percentage changes and of absolute values at different times and under the various experimental conditions were compared by Student's t test. Differences between means were considered significant if PG0.05. In addition coefficients of correlation ( r ) between absolute values of IDT and FC with time and against adequate controls were also calculated for some experimental groups and if significant, presented with their corresponding curves.
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In-vitro efect
of
chagasic sera
on rat atrium
60 min these parameters showed a similar and progressive decrease in all cases. Due to the stability problems during the first 60 min (see methods), the rather scattered absolute values observed at this time (see Fig. 2), and the fact that the effect of chagasic sera appears to be a relatively slow developing process, it was decided to express the results not only as percentage changes at 120 and 180min against controls taken at 60 min, but also to obtain coefficients of correlation between the absolute magnitudes of tension and frequency with time (at 120 and 180 min versus 60 min; see methods). Figure 1 plots the percentage changes in FC against time, corresponding to atria immersed in different media. Fig. 2 depicts absolute magnitudes of frequency versus time as a curvilinear relationship for the same experiments; it also
shows the coefficient ofcorrelations and indicates the levels of significance with the corresponding P values. It is clear that both presentations of the experimental results show that atria beating in KRB, in K R B plus NHS, or in K R B plusEVI(-)S, react with a progressive reduction of FC as time goes on, while the frequency of atria immersed in KRB plus EVI( i - ) S augmented significantly with time.
IX ::
.
.
I -
I20
180
2 Relationship between absolute magnitudes of the frequency of contractions with respect to time, of isolated rat atria suspended in different media. FIG.
-20
60
I20
180
Time ( m i d
1 Influence of diferent suspending media on the frequency of contractions ( F C ) of isolated rat atria. Curves show relative changes (%) during I20 min. This percentage was calculated by comparison against absolute values registered at 60 min of activity and considered as controls (see methods). Total time of atrial activity = 180 min. KRB = Krebs-Ringerbicarbonate; NHS == normal human sera; EVI(-)S = EVI negative chagasic human sera; EVI(+)S = E VI positive chagusic human sera. Numbers in parentheses refer to the number of preparations. Vertical bars indicate the S E M . Note a progressive increment of F C only in atria suspended in KRBplus EVI( +)S when compared with the three other groups ( P gO.001). FIG.
Curves show absolute changes (mg) during 120 min. Changes were plotted against absolute values recorded at 60min of activity and considered as controls (see methods), Total time of atrial activity = 180 min. 0 = individual absolute magnitudes of each atrial preparation. (Other conditions, details, and abbreviations as in Fig. 1.) Note the statistically significant increment of atrial FC beating in K R B plus EVI( + ) S and the significative reduction when suspended in the other three solutions.
EVI( + ) S : y = -0.004x2+1.02x +115.6; r 0.28; PcO.02 (for 72 df). E V I ( - ) S : y = 0.003x2-0.7x+178; r = 0.43; P ~ 0 . 0 2(for 72 df). NHS: y = 0 . 0 0 3 ~ ~ 4 l . 8 7 187.5; .~ r = 0.42; PgO.001 (for 81 df). 0.45; K R B : y = 0.002x2-0.667~+181.2; r PdO.001 (for 72 d f l .
+
I"
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Q 0 60
617 Sterin-Borda, Cossio, M . Gimeno, A . Gimeno, Diez, Laguens, Meckert, and Arana
An identical analysis of the results obtained regarding the IDT show only a small but not significant change in all the four suspending media. The percentage changes are illustrated in Fig. 3. Furthermore, the coefficients of correlation for absolute values of IDT with time (at 120 and 180min) against controls recorded at 60 min are: 0.07 (NS for 72 df) in KRB; 0.1 1 (NS for 81 df) in KRB plus NHS; 0.05 (NS for 27 df) in KRB plus EVI(-)S, and 0.16 (NS for 72 df) in KRB plus EVI( +)S. The increment of the FC observed in the presence of EVI( +)S, was prevented by the addition of MJ-1999 to the suspending solution; but neither phentolamine nor cyproheptadine were able to modify the positive chronotropic effect
associated with the presence of EVI ( + )chagasic serum (Fig. 4). All these agents were added at 60 minutes. 1
1
+'O1
0 Time
-20
L
p
bO
I20
I80
Time ( m i d
.3
Influence of different media on the isometric developed tension ( I D T ) of isolated rat atria. Conditions, details, and abbreviations as in Fig. 1. Note a small and not statistically significant change similar in all the groups (for coeficients of correlution of absolute values see text). FIG
(mid
60
I
180
I20
F I G . 4 Effects of MJ-1999: phentolarnine, or cyproheptadine on the frequency of contractions of isolated rat atria suspended in EVI( +) chagasic sera. Curves show percentage changes during 180 min. Percentages were calculated by comparison against absolute initial values taken as controls arid recorded 10 min following isolation, ie, at 0 time in the figure (see methods). Abbreviations for sera and other details are the same as in Fig. 1. Note that the incubation with MJ-1999, but neither with phentolamine (Phen), nor with cyproheptadine (Cyp), prevent the increment of frequency induced by the presence of EVI( + ) S . All the agents mentioned were added at 60 rnin.
TABLE 2 Values (means S E M ) of isometric developed tension ( I D T ) and frequency of contraction ( F C ) of isolated rat atria immersed in different suspending media*
Suspending media KRB
K R B plus NHS
K R B plus EVI(-) rhagasic sera
KRB plus EVI( +) chogasic sera
480.0 i 5 9 . 2 (n=6) 146.012.0 (n = 6)
467.7 140.3 (n-9) 174.4f9.4 (n -9)
_______-__________ IDT (ms)
FC (beats/min)
510.0 ' 74.0 ( n - 8) 137.516.5 (n - 8)
420.0 +33.8 (n= 10) 134.014.2 (n -10)
*Absolute values recorded at 180 min of activity (see methods).
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1
-30
618
hi-vitro efect of chagasic sera
012
rat atrium
ig,
I50
0 IDT IFC
I2 5
5 Effect of norepinephrine over the isometric developed tension ( I D T ) and the frequency of contractions ( F C ) of isolated rat atria. The columns show maximal percentage of changes recorded at 2-3min following the addition of norepinephrine as compared with values obtained at 180 min and shown in Table 2. In all cases the neurotransmitter was added immediately after 180 min (see methods). Dots above columns indicate the SEM. Other symbols, abbreviations and details are as in Fig. 1. Note that the enhancing effect of added norepinephrine upon IDT and FC is significantly smaller over atria suspended in a medium with EVI-( +)S. FIG.
-$
100
10)
I
15 0
6 50
25
C
i
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F I G . 6 (A) Direct immunofluorescence microphotograph of an atrial section which has been beating in medium with an EVI( t) chagasic serum; a fluorescein-labelled goat anti-human gamma globulin antiserum was used. Positive staining of two sarcolemmal areas is seen as well as a place in which fibres contract ( x 4000.) (B)Phase contrast microphotograph of the same area shown in Fig. 6 A ( x 4000.)
619
Sterin-Borda, Cossio, M . Gimeno, A . Gimeno, Diez, Laguens, Meckert, and Arana
F I G . I Electronmicrograph of the same atrial preparation of Fig. 6a, treated with peroxidaselabelled, goat gamma globulin anti-human gamma globulin, followed by histochemical staining of peroxidase activity. An electron dense reaction in the plasma membrane of a working myocardial cell is observed. ( x I4 000.)
Structural alterations The ultrastructural study of the controls showed that the atrial cardiocytes presented a normal Immunofluorescent studies appearance on morphological basis. No ultraDeposits of immunoglobulins and C3 were structural evidence of hypoxia such as mitoobserved in the atria which contracted in medium chondrial swelling was seen. In the atria incubated with EVT positive sera containing EVI( +) sera. They were located in the sarcolemmal area (Fig. 6A), and although peculiar modification of the sarcolemma and of the deposits were focal, they were widely the peripheral sarcoplasm was seen. In the latter, distributed. In the atria which were beating in flattened cisternae were seen immediately beneath the plasma membrane. In some areas it was the other media no staining was observed. hard to distinguish between both structures and the plasma membrane appeared triple layered Ultrastructural immunochemical studies (Fig. 8) Deposits of gamma globulin were seen in the At places the plasma membranes of adjacent plasma membrane of working myocardial cells muscle cells presented a peculiar appearance and vascular endothelial cells of the atria which consisting of numerous whorls which formed a were beating with the EVI(-t) sera (Fig. 7). convoluted structure in which it was hard to ‘Blocking’ experiments performed applying the distinguish individual plasma membranes (Fig. unlabelled antibody previously to the treatment 9). with the labelled one abolished the reaction; As pilot observations suggested that atria negative results were obtained with the peroxi- beating in EVI(+) sera showed an increased dase-labelled rabbit-anti-rat IgG antibody, or number of atrial granules, a morphometric study of these structures was carried out. No with peroxidase alone. Negative results were obtained with the atria difference of the granule mass and number could which beat with KRB alone or with EVI(-) sera be found between the control and the experimental group. In the former 3.9% of atrial added or with fresh NHS.
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Effect of norepinephrine upon isolated atrial preparations suspended in different media A final experimental series was performed in a third group of atria. They were allowed to function during 3 h in all the supporting solutions described above, before being exposed to exogenous norepinephrine. The magnitudes of IDT and FC (in absolute values) were recorded at the end of 180 min of activity (see Table 2). As can be seen, the IDT has a similar magnitude in all the suspending solutions. However, the FC showed values significantly higher in KRB plus EVI(+) chagasic sera than in the other three media; this being in keeping with the findings described in the larger group of atria mentioned in the previous section. The addition of norepinephrine to atria immersed in KRB, in KRB plus NHS, or in KRB plus EVI(-) chagasic sera, elicited a comparable enhancement of both IDT and FC (Fig. 5). On the contrary, atria suspended in KRB plus EVI(+) chagasic sera (Fig. 5 ) responded to added norepinephrine with a significantly smaller change of both developed tension and frequency of beatings (IDT: Pt0.01;FC: PtO.O1).
620
In-vitro efect of chagasic sera
011
rat atrium
cytoplasm was represented by atrial granules and in the atria incubated with EVI(+) sera, the granules composed 4.2 % of the cytoplasm. Discussion In the present report, employing an hyperacute experimental design, the ability of EVI antibody to interact with the plasma membrane of living myocardial cells could be demonstrated. In addition, morphological alterations appeared in the sarcolemma of the cells in which the antibody is fixed, suggesting that these alterations are related to an action of the EVI antibody. Although bound p1 C-A was also demonstrated, classical holes as induced by the C system in other membranes (Humphrey and Dourmashkin, 1965; Anziano et al, 1972) were not observed.
The present study demonstrates that EVI positive sera can induce functional modifications in isolated atrial preparations. After brief exposures of 60 min, inotropic and chronotropic behaviour remained similar to those of EVI negative sera and controls, suggesting that the changes observed thereafter are not related to the presence of higher concentrations of pharmacologically active hormones or substances. Instead, the slow onset of the observed changes suggest a delay due to the progressive fixation of the antibodies to the membranes. In the case of cardiac anaphylaxis with isolated preparations (Feigen and Prager, 1969), histamine release and its effects on frequency and tension were observed within a few minutes. Although EVI antibodies in chronic chagasic cases are mainly IgG, so that anaphylaxis might be expected
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F I G . 8 High power view of two atrial muscle cells sarcolemma. Immediately beneath the plasma membrarte flattened cisternae can be seen (arrows). ( x 44 000.)
621 Sferin-Borda, Cossio, M . Gimeno, A . Citneno, Diez, Laguens, Meckert, and Arana
. 9 Electronmicrograph of two atrial cardiocytes. The arrow points to an area where adjacent plasmalemmae show a highly convolirted appeararice. FIG
through the activation of the complement system (Dias Da Silva and Lepow, 1967; Jensen, 1967; Miiller-Eberhard, 1968), the effects observed in the present study are not likely to be due to significant amounts of histamine release because of the slow onset of the changes. In addition, cyproheptadine did not alter the effect of EVI(+) chagasic sera. In atria beating in the media containing EVI( +) sera, a significantly increased frequency of beating was observed at 120 and 180min, whereas in those beating in media containing EVI(-) sera or in the controls the frequency diminished. This increase in frequency could be prevented by a p-adrenergic blocking agent, but not by an a-adrenergic blocking agent, or by an anti-histaminic compound. Two explanations are possible: (1) the interaction of the antibody with the specific antigen induces a slow liberation of endogeneous norepinephrine ; (2) the fixation of the EVI antibody induces a partial ‘agonist like’ effect through the Padrenergic receptor which appears to be located in the plasma membrane (Lefkowitz, 1973). The finding that tension was not substantially
modified by EVI( i - )sera remains unexplained. Both tension and frequency response changed in parallel when exogenous norepinephrine was added to the medium containing EVI( +) sera, suggesting that the antibody also had at least some effect on tension. However, at present, it is not easy to ascertain whether the smaller percentage of changes of FC following norepinephrine, observed in atria subjected to the presence of EVI(+) sera, is due to a diminished influenced of the neurotransmitter over pacemaker cells, directly associated to the antibody, or to the result that control frequency values before norepinephrine were not already higher in this group. However, this is not the case regarding the smaller inotropic reactivity towards this agent because control magnitudes of IDT before its addition were similar. Therefore, it would appear to support the existence of an effect of the antibody upon atrial peak tension, at least with respect to the effect of exogenous norepinephrine. The present results may be explained if the EVI antibody acted as a partial p-adrenergic agonist that could both increase frequency beating and also to some extent block
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( x 16 000.)
622 In-vitro effect of chagasic sera on rat atrium
The authors wish to express their thanks to Dr Arnold M. Katz and Dr Deborah Doniach for their advice and for revising and improving the English manuscript. The valuable technical assistance of M. B. Casanova is gratefully acknowledged.
References Anziano, D. F., Dalmasso, A. P., Lelchuck, R., and Vasquez, C. (1972). Role of complement in immune lysis of Tr.vpononosoma cruzi. Infection and Imnriinity. 6 , 860-865. Cossio, P. M., Diez, C., Szarfman, A,. Kreutzer. E., Candi010, B., and Arana, R. M. (1974a). Chagasic cardiopalhy: demonstration of a serum gamma globulin factor which reacts with endocardium and vascular structures. Circiclotion. 49, 13-2 I. Cossio, P. M., Laguens, R. P., Diez, C., Szarfman, A,, Segal, A., and Arana, R. M. (1974b). Chagasic cardiopathy: antibodies reacting with plasma membrane of striated muscle and endothelial cells. Cirrulation, 50, 1252-1259. Dias Da Silva, W., and Lepow, I. H. (1967). Complement as a mediator of inflammation. 11. Biological properties of anaphylatoxin prepared with purified components of human complement. Journal of Experinientd Medicine, 125, 921-946. Gimeno, A. L., Gimeno, M. F., and Webb, J . L. (1963). Action of sex steroids on the electrical and mechanical properties of rat atrium. American Journal of Plr.vsiologjt. 205, 198- 199. Feigen, G. A., and Prager, D. J. (1969). Experimental cardiac anaphylaxis: physiologic, pharmacologic and biochemical aspects of immune reactions in the isolated heart. American Journal of Cardiology, 24, 4 7 4 4 9 1 . Hall, R., Smith, B. R., and Mukhtar, E. D. (1975). Thyroid stimulators in health and disease. Clinical Enclorrinolog?: 4, 213-230. Humphrey, J . H., and Dourmashkin, R. R. (1965). Electron microscope studies of immune cell lysis. I n Complenient: A Ciba Foiindation Synipsoiuin, ed. by G . E. W. Wolstenholme and J. Knight, pp. 175-186. Churchill: London. Jensen, J. (1967). Anaphylatoxin and its relation to the complement system. Science. 155, I 122- I 123. Kabat, E. A., and Mayer, M. M. (1964). Experinientcil Imnrunochemistry, p. 135. Thomas: Springfield, Illinois. Kaplan, M. H. (1969). Autoimmunity to heart. In Textbook qflmnrunopathology.ed, by P. Miescher and H. J. MullerEberhard, vol. 2, pp. 647-650. Grune and Stratton: New York and London. Lacuara, J. L., Gimeno, A. L., and Gimeno, M. F. (1971). Effects of monosacharides, pyruvate, acetate and butyrate on the developed tension and ATP levels in of hypodynamic isolated rat atria. Proceedings of the Society for Experimental Biology and Medicine. 136, 1369-1 373. Laguens, R. P. (1971). Morphometric study of myocardial mitochondria in the rat. Joiirnal of Cell Biology. 48, 673675. Lancet (1974). The latest on LATS. Lancet, 2, 4 4 3 4 4 4 . Lefkowitz, R. J. (1973). Isolated beta-adrenergic binding si!es. A potential assay vehicle for catecholaniines. Pharniacological Reviews. 25, 259-267. Miiller-Eberhard, H. J. (1968). The serum complement system. In Textbook of Iminunopattiology, ed. by P. Miescher and H. J. Mu!ler-Eberhard, pp. 3 9 4 7 . Grune and Stratton: New YJrk and London. Smith, B. R., and Hall, R. (1974). Thyroid-stimulating immunoglobulins in Graves’ disease. Lancet. 2, 4 2 7 4 3 0 . Smith, T. W., Wagner, H., Young, M., and Kyte, J. (1973). Effects of antibodies specifically directed against N a ’ , K’ATPase. Journal of Clinical Investigation, 52, 78a. Vurek, G. G., Prager, D. J., and Feigen, G . A. (1967). Antibody concentration and temperature as determinants of in vitro sensitization and histamine release in isolated cardiac tissues. Joirrnal of Immunology. 99, 1243-1253.
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the response of the p-receptors to exogenous norepinephrine. A similar pattern of activation of biological mechanisms has been described in the case of LATS which is acting through the reaction of antibodies with membrane TSH receptors (Lancet, 1974; Smith and Hall, 1974). In this case, the evidence was obtained by a competition radioimmunoassay (Hall et a/, 1975). The EVI antibody, also reacting with plasma membranes of striated muscle (Cossio er a/, 1974a), does not have species-specificity (Cossio et a/, 1974b) and has biological activity. It cannot be established whether the EVI antibody acts directly through a specific reaction with antigenic determinants related to the p-adrenergic receptor itself, or, whether these effects arise from indirect mechanisms due to an interaction with the plasma membrane of the myocardial cells. The present report demonstrates that EVI antibody can interact with living myocardial cells to induce both morphological and functional changes. These effects appear to be related predominantly to the plasma membrane of myocardial cells. This finding is in accordance with the physiological properties of this structure, and supports the hypothetical participation of EVI antibody in some of the pathogenetic mechanisms of Chagas’ heart disease (Cossio et a / , 1974a; 1974b). Many forms of arrythmias and puzzling responses to antiarrythmic drugs and therapeutic electrical procedures are commonly found in Chagas’ heart disease; sudden cardiac death is also prominent. However, further data will be needed before the functional, pharmacological, and morphological modifications observed with the EVI antibody in our hyperacute in-vitro model can be related to the slowly developing rhythm and conduction disturbances observed in the chronic heart involvement of American trypanosomiasis.
Effect of chagasic sera on the rat isolated atrial preparation: immunological, morphological and functional aspects1 LEONOR S T E R I N - B O R D A , P A T R I C I O M. COSSIO, MARTHA F . G I M E N O , ALVARO L . GIMENO, CARLOS D I E Z , R U B E N P . L A G U E N S , P A T R I C I A C A B E Z A MECKERT, and R O B E R T O M . A R A N A
From the Centro de Estudios Farmacoldgicos y de Principios Naturales, and Instituto de Neurobiologia (Consejo Nacional de Investigaciones CientSficas y Tkcnicas de la Repfiblica Argentina - CONICET -), Buenos Aires, the Laboratory of Rheumatology and Immunology and the Cardiology Unit, CEMIC, Buenos Aires, and the Chtedra de Patologia II, Facultad de Medicina de La Plata, La Plata. Argentina
This work was supported in part by grants from Fundacibn Cossio, ADAR, Academia Nacional de Medicina de Buenos Aires, and CONICET (Grants 6638 and 5863/a). L.S.B.and P.C.M. are Advanced Research Fellows and M. F. G . , A.L.G.,and R.M.A. are Senior Investigators of the CONICET. Reprint requests to R.M.A., CEMIC, Sanchez de Bustamante 2560, Buenos Aires, Argentina.
The presence of an antibody reacting with and striated muscle (EVI antibody) has been described in Chagas’ disease (cOssiO et 974a). In a more recent report, it was shown that the EVI antibody reacts with antigenic determinants located in
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A U T H O R S ’ S Y N O P S I S An antibody reacting with the plasma membrane of working myocardial cells, skeletal muscle fibres, and endothelial cells (EVI antibody) has been described in the sera of patients with Chagas’ disease. In the present study of rat isolated atrial preparations beating in different media, direct immunofluorescence and ultrastructural immunohistochemical procedures indicate that the antibody can interact with the living tissue, becoming fixed to the plasma membranes. Transmission electronmicroscopy studies also showed the presence of sarcolemmal alterations. These observations suggest a possible pathogenic effect of the EVI antibody. The presence of EVI-positive sera in the beating medium leads to a significant increase in the frequency of contractions ; no significant effects of EVI-positive sera in contractile force were seen. The increase in frequency could be prevented by previous treatment with a P-adrenergic blocking agent (MJ-1999), but not by an a-blocker (phentolamine) or by an anti-histamine compound (cyproheptadine). The changes described were observed only in those atrial preparations which were beating in media containing EVI-positive sera. In those atria beating in control media (KR, KR plus normal human serum, KR plus EVI-negative chagasic serum), neither immunological nor morphological or functional changes were seen. In addition, some observations suggest that the presence of EVIpositive chagasic serum diminished atrial stimulation after added norepinephrine. These results suggest the possibility that the EVI antibody may act as a P-adrenergic agonist at the cell plasma membrane level. Such an effect might account for some of the clinical features of chronic Chagas’ heart disease.
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(KRB) (Gimeno et al, 1963) kept at room temperathe plasma membrane of working myocardial ture. The substrate for this medium was glucose at cells, skeletal muscle fibres, and vascular endo- 5.5 mmol/litre. It was gassed with a mixture of 95"; O2and 5% CO,. The atria were then separated from thelial cells (Cossio et a/, 1974b). Although it is the ventricles, carefully dissected, attached to a reasonable to assume that the plasma membranes glass holder, and immersed in a tissue chamber of different cells are, in general, able to interact filled with different nutrient fluids: ( I ) 20ml of in vivo with circulating antibodies, it has been KRB solution; (2) 17 ml of KRB plus 3 ml of fresh observed in some experimental models that normal human serum (NHS); (3) 17ml of KRB heart muscle cells did not easily react in vivo plus 3 ml of fresh chagasic EVI(-) human serum with specific antibodies (Kaplan, 1969). Further- (EVI(-)S); (4) 17 ml of KRB plus 3 ml of fresh more, a recent report showed that antibodies chagasic EVI( +) human serum (EVI( +)S). The against the Na+, K+-activated ATPase inhibit tissue bath solutions were gassed with a mixture of the enzyme activity in extracted organs, but not 95% 0, and 5% C O , throughout the experiments. Temperature and pH were 30" and 7.4. In each in the intact cell, suggesting that the antigenic bath solution 0.1 ml of Antifoam C (Sigma Chemical determinant(s) of this enzyme may be masked at Co) was added. This agent did not modify any of the the surface of the intact cells (Smith et a/, 1973). measured parameters of the beating atria. A resting The present study was performed (1) to in- tension of 750 mg was applied to the atria by means vestigate the ability of the EVI antibody to inter- of a micrometric device and their developed tension act with living myocardial cells and (2) if this and frequency were recorded by means of a force was so, to observe if this reaction could alter displacement transducer' coupled to an ink writing their physiological behaviour or modify the well- oscillograph'. Under the above procedure the isoknown actions of some hormones and drugs. The lated rat atria contracted in almost nearly isometric isolated atrial preparation was selected as the conditions. The peak tension developed by atria, over the experimental model because experimental card- resting applied tension, and their rate of contraciac anaphylaxis has been extensively studied in tion were recorded by taking tracings of the isothis model (Feigen and Prager, 1969) and also metric developed tension (IDT) and of the frebecause the atrium and the region which includes quency of contractions (FC), as previously reported the sinoatrial node is the part of the heart most (Lacuara et al, 1971). The mean absolute magnitudes of IDT and FC observed at 10 min following responsive to aoaphylaxis (Vurek et a/, 1967). isolation were taken as the initial control values (0 time). The IDT was determined in mg and the FC Material and methods measured by counting the number of atrial beats Sera per min. The preparations were then observed for Sera were obtained from 15 EVI( +) and six EVI(-) functional variations with respect to time, selecting chagasic individuals and from 10 blood donors. for recordings changes at 60, 120, and 180 min. In The EVI positivity or negativity of sera was deter- some cases the modifications observed were exmined by means of the indirect immunofluorescence pressed as the percentage of change from the level technique (Cossio et al, 1974a). All positive sera had of initial control values. However, in as much as an EVI titre 21/80, Sera were immediately used, or the first 60 min of activity might have some degree kept frozen at -7O"C, for periods up to 7 d. Comple- of spontaneous lack of stability, arising from the ment haemolytic activity of sera (CH50) was deter- trauma imposed by the dissection and the mounting mined in each sample by the method of Kabat and procedures, the absolute magnitudes of IDT and Mayer (1964) and all contained >35 CH50 units/ml FC recorded at 60 min were taken as a second (normal values 25-50 CH50 unitslml). The subjects control value and used to make the percentage and had not received any drug or therapeutic agent for absolute comparisons with records obtained at 120 the previous month. and 180 minutes. The influence on IDT and FC, elicited by noreIsolated atrial preparations pinephrine (Sigma Chemical Co), MJ-1999 (SotaMale albino rats of the Wistar strain weighing lol@, Mead Johnson), phentolamine (RegitineQ, between 200-250g were used. The animals were Ciba), or cyproheptadine (PeriactinQ, Merck, sacrificed by decapitation and the entire heart was Sharp and Dohme) was also studied over atria quickly excised and placed in a Petri dish filled with Statham, UC3-Gold Cell. a modified Krebs-Ringer-bicarbonate solution * Beckrnan, Type S-11 Dynograph.
615 Sterin-Borda, Cossio, M . Gimeno, A . Gimeno, Diez, Laguens, Meckert, and Arana overnight in 0.1 mol/litre phosphate buffer pH 7.3 and sectioned at 1 0 0 ~with a freezing microtome. The sections were treated (at 4°C) in one of the following ways: ( I ) incubation for 3 d with goat gamma globulin anti-human globulin, labelled with peroxidase; (2) incubation with rabbit gamma globulin anti-rat IgG (absorbed with normal human serum), labelled with peroxidase (1.2 mg/ml in PBS); (3) as ( I ) but previously blocked with unlabelled goat gamma globulin anti-human immunoglobulins. The material was processed as previously described (Cossio et a / , 1974b) and examined with a Philips transmission electron-microscope operated at 60 kV. The same batch of goat anti-human gamma globulin anti-serum employed for the immunofluorescence studies was used; the globulin fraction of the antiserum was obtained and IabelJed with horseradish peroxidase as reported elsewhere (Cossio et al. 1974b). Ultrastructirral microscopy Glutaraldehyde-fixed material was conventionally processed and examined by transmission electronmicroscopy. In the tissue sections morphometric study of the atrial granules was performed as previously described (Laguens, 1971).
Tissue studies Immediately after the experiments, the atria were immersed in 150ml of phosphate buffered saline (PBS) at 22°C. The atria were allowed to beat for I to 2 min after which they were washed several times for 5 min with PBS. The material was then divided into three pieces; one was immediately frozen at -2O"C, the second was fixed for 4 h at 4°C in 40/, formaldehyde in phosphate buffer 0.1 mol/litre, pH 7.3, and the third was fixed in 4q4, glutaraldehyde in phosphate buffer.
Direct immunofluorescence Cryotstat sections, 2 p thick, were cut from the frozen tissue and stained with fluorescein-labelled goat gamma globulins anti-human immuniglobulins (Cossio et al, 1974a), and anti-human C3 (Hyland Travenol Lab, Los Angeles, California, USA), as previously described (Cossio et al, 1974a).The tissues were examined with a Carl Zeiss microscope (Federal Republic of Germany) fitted with epicondenser fluorescence equipment and a phase contrast condenser.
Ultrastructural immunohistochemical procedure The atrial pieces fixed in formaldehyde were washed
Results Functional characteristics of isolated rat atrial preparations suspended in KRB, in NHS, and in EVI(-) or EVI( +) chagasic sera Table I shows that the absolute initial values (0 time) of IDT or FC of atria suspended in different media have similar magnitudes. At TABLE 1
fnitial values (means i SEM) of isometric developed tension (IDT) and frequency of contraction (FC) of isolated rat atria immersed in different suspending media* ID T
FC
__
(mgl
(beafslmin)
KRB
390 139.5 (n -25) 301 135. I (n=24) 378 1 5 3 . 0 (n=- 1 I ) 326 126.3 (n: 25)
I70 1 4 . 6 (n = 25) 175 17.1 (n = 24) 162 1 9 . 6 (n= 11) 175 +5.2 (n = 25)
Suspending media
KRB plus NHS KRB PIUS EVI (-) chagasic sera KRB plus EVl ( +) chagasic sera
*Absolute initial values recorded at LO min following isolation (0 time, see methods).
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beating in the different suspension media. Their final concentration in the tissue bath solution were: 3 . 9 lo-*, ~ 0.1, 1 . 6 ~ and 7 x 10--5mmol/ml, respectively. Freshly prepared solutions of these drugs were added to the suspending medium in a volume not higher than 0.2 ml. The addition of all these agents, except norepinephrine, was always performed at 60rnin of activity. For the experiments testing the effects of norepinephrine, a second group of atria was used. They were allowed to beat during 180min in the different media and the absolute magnitudes of IDT and FC were then recorded. Immediately a submaximal concentration of the drug was added to the bath solution, its effects registered during 10 min, and the maximal response (occurring most often at 2 or 3 min) measured, and expressed as a percentage change against the 180 min values. Results of percentage changes and of absolute values at different times and under the various experimental conditions were compared by Student's t test. Differences between means were considered significant if PG0.05. In addition coefficients of correlation ( r ) between absolute values of IDT and FC with time and against adequate controls were also calculated for some experimental groups and if significant, presented with their corresponding curves.
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of
chagasic sera
on rat atrium
60 min these parameters showed a similar and progressive decrease in all cases. Due to the stability problems during the first 60 min (see methods), the rather scattered absolute values observed at this time (see Fig. 2), and the fact that the effect of chagasic sera appears to be a relatively slow developing process, it was decided to express the results not only as percentage changes at 120 and 180min against controls taken at 60 min, but also to obtain coefficients of correlation between the absolute magnitudes of tension and frequency with time (at 120 and 180 min versus 60 min; see methods). Figure 1 plots the percentage changes in FC against time, corresponding to atria immersed in different media. Fig. 2 depicts absolute magnitudes of frequency versus time as a curvilinear relationship for the same experiments; it also
shows the coefficient ofcorrelations and indicates the levels of significance with the corresponding P values. It is clear that both presentations of the experimental results show that atria beating in KRB, in K R B plus NHS, or in K R B plusEVI(-)S, react with a progressive reduction of FC as time goes on, while the frequency of atria immersed in KRB plus EVI( i - ) S augmented significantly with time.
IX ::
.
.
I -
I20
180
2 Relationship between absolute magnitudes of the frequency of contractions with respect to time, of isolated rat atria suspended in different media. FIG.
-20
60
I20
180
Time ( m i d
1 Influence of diferent suspending media on the frequency of contractions ( F C ) of isolated rat atria. Curves show relative changes (%) during I20 min. This percentage was calculated by comparison against absolute values registered at 60 min of activity and considered as controls (see methods). Total time of atrial activity = 180 min. KRB = Krebs-Ringerbicarbonate; NHS == normal human sera; EVI(-)S = EVI negative chagasic human sera; EVI(+)S = E VI positive chagusic human sera. Numbers in parentheses refer to the number of preparations. Vertical bars indicate the S E M . Note a progressive increment of F C only in atria suspended in KRBplus EVI( +)S when compared with the three other groups ( P gO.001). FIG.
Curves show absolute changes (mg) during 120 min. Changes were plotted against absolute values recorded at 60min of activity and considered as controls (see methods), Total time of atrial activity = 180 min. 0 = individual absolute magnitudes of each atrial preparation. (Other conditions, details, and abbreviations as in Fig. 1.) Note the statistically significant increment of atrial FC beating in K R B plus EVI( + ) S and the significative reduction when suspended in the other three solutions.
EVI( + ) S : y = -0.004x2+1.02x +115.6; r 0.28; PcO.02 (for 72 df). E V I ( - ) S : y = 0.003x2-0.7x+178; r = 0.43; P ~ 0 . 0 2(for 72 df). NHS: y = 0 . 0 0 3 ~ ~ 4 l . 8 7 187.5; .~ r = 0.42; PgO.001 (for 81 df). 0.45; K R B : y = 0.002x2-0.667~+181.2; r PdO.001 (for 72 d f l .
+
I"
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Q 0 60
617 Sterin-Borda, Cossio, M . Gimeno, A . Gimeno, Diez, Laguens, Meckert, and Arana
An identical analysis of the results obtained regarding the IDT show only a small but not significant change in all the four suspending media. The percentage changes are illustrated in Fig. 3. Furthermore, the coefficients of correlation for absolute values of IDT with time (at 120 and 180min) against controls recorded at 60 min are: 0.07 (NS for 72 df) in KRB; 0.1 1 (NS for 81 df) in KRB plus NHS; 0.05 (NS for 27 df) in KRB plus EVI(-)S, and 0.16 (NS for 72 df) in KRB plus EVI( +)S. The increment of the FC observed in the presence of EVI( +)S, was prevented by the addition of MJ-1999 to the suspending solution; but neither phentolamine nor cyproheptadine were able to modify the positive chronotropic effect
associated with the presence of EVI ( + )chagasic serum (Fig. 4). All these agents were added at 60 minutes. 1
1
+'O1
0 Time
-20
L
p
bO
I20
I80
Time ( m i d
.3
Influence of different media on the isometric developed tension ( I D T ) of isolated rat atria. Conditions, details, and abbreviations as in Fig. 1. Note a small and not statistically significant change similar in all the groups (for coeficients of correlution of absolute values see text). FIG
(mid
60
I
180
I20
F I G . 4 Effects of MJ-1999: phentolarnine, or cyproheptadine on the frequency of contractions of isolated rat atria suspended in EVI( +) chagasic sera. Curves show percentage changes during 180 min. Percentages were calculated by comparison against absolute initial values taken as controls arid recorded 10 min following isolation, ie, at 0 time in the figure (see methods). Abbreviations for sera and other details are the same as in Fig. 1. Note that the incubation with MJ-1999, but neither with phentolamine (Phen), nor with cyproheptadine (Cyp), prevent the increment of frequency induced by the presence of EVI( + ) S . All the agents mentioned were added at 60 rnin.
TABLE 2 Values (means S E M ) of isometric developed tension ( I D T ) and frequency of contraction ( F C ) of isolated rat atria immersed in different suspending media*
Suspending media KRB
K R B plus NHS
K R B plus EVI(-) rhagasic sera
KRB plus EVI( +) chogasic sera
480.0 i 5 9 . 2 (n=6) 146.012.0 (n = 6)
467.7 140.3 (n-9) 174.4f9.4 (n -9)
_______-__________ IDT (ms)
FC (beats/min)
510.0 ' 74.0 ( n - 8) 137.516.5 (n - 8)
420.0 +33.8 (n= 10) 134.014.2 (n -10)
*Absolute values recorded at 180 min of activity (see methods).
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1
-30
618
hi-vitro efect of chagasic sera
012
rat atrium
ig,
I50
0 IDT IFC
I2 5
5 Effect of norepinephrine over the isometric developed tension ( I D T ) and the frequency of contractions ( F C ) of isolated rat atria. The columns show maximal percentage of changes recorded at 2-3min following the addition of norepinephrine as compared with values obtained at 180 min and shown in Table 2. In all cases the neurotransmitter was added immediately after 180 min (see methods). Dots above columns indicate the SEM. Other symbols, abbreviations and details are as in Fig. 1. Note that the enhancing effect of added norepinephrine upon IDT and FC is significantly smaller over atria suspended in a medium with EVI-( +)S. FIG.
-$
100
10)
I
15 0
6 50
25
C
i
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F I G . 6 (A) Direct immunofluorescence microphotograph of an atrial section which has been beating in medium with an EVI( t) chagasic serum; a fluorescein-labelled goat anti-human gamma globulin antiserum was used. Positive staining of two sarcolemmal areas is seen as well as a place in which fibres contract ( x 4000.) (B)Phase contrast microphotograph of the same area shown in Fig. 6 A ( x 4000.)
619
Sterin-Borda, Cossio, M . Gimeno, A . Gimeno, Diez, Laguens, Meckert, and Arana
F I G . I Electronmicrograph of the same atrial preparation of Fig. 6a, treated with peroxidaselabelled, goat gamma globulin anti-human gamma globulin, followed by histochemical staining of peroxidase activity. An electron dense reaction in the plasma membrane of a working myocardial cell is observed. ( x I4 000.)
Structural alterations The ultrastructural study of the controls showed that the atrial cardiocytes presented a normal Immunofluorescent studies appearance on morphological basis. No ultraDeposits of immunoglobulins and C3 were structural evidence of hypoxia such as mitoobserved in the atria which contracted in medium chondrial swelling was seen. In the atria incubated with EVT positive sera containing EVI( +) sera. They were located in the sarcolemmal area (Fig. 6A), and although peculiar modification of the sarcolemma and of the deposits were focal, they were widely the peripheral sarcoplasm was seen. In the latter, distributed. In the atria which were beating in flattened cisternae were seen immediately beneath the plasma membrane. In some areas it was the other media no staining was observed. hard to distinguish between both structures and the plasma membrane appeared triple layered Ultrastructural immunochemical studies (Fig. 8) Deposits of gamma globulin were seen in the At places the plasma membranes of adjacent plasma membrane of working myocardial cells muscle cells presented a peculiar appearance and vascular endothelial cells of the atria which consisting of numerous whorls which formed a were beating with the EVI(-t) sera (Fig. 7). convoluted structure in which it was hard to ‘Blocking’ experiments performed applying the distinguish individual plasma membranes (Fig. unlabelled antibody previously to the treatment 9). with the labelled one abolished the reaction; As pilot observations suggested that atria negative results were obtained with the peroxi- beating in EVI(+) sera showed an increased dase-labelled rabbit-anti-rat IgG antibody, or number of atrial granules, a morphometric study of these structures was carried out. No with peroxidase alone. Negative results were obtained with the atria difference of the granule mass and number could which beat with KRB alone or with EVI(-) sera be found between the control and the experimental group. In the former 3.9% of atrial added or with fresh NHS.
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Effect of norepinephrine upon isolated atrial preparations suspended in different media A final experimental series was performed in a third group of atria. They were allowed to function during 3 h in all the supporting solutions described above, before being exposed to exogenous norepinephrine. The magnitudes of IDT and FC (in absolute values) were recorded at the end of 180 min of activity (see Table 2). As can be seen, the IDT has a similar magnitude in all the suspending solutions. However, the FC showed values significantly higher in KRB plus EVI(+) chagasic sera than in the other three media; this being in keeping with the findings described in the larger group of atria mentioned in the previous section. The addition of norepinephrine to atria immersed in KRB, in KRB plus NHS, or in KRB plus EVI(-) chagasic sera, elicited a comparable enhancement of both IDT and FC (Fig. 5). On the contrary, atria suspended in KRB plus EVI(+) chagasic sera (Fig. 5 ) responded to added norepinephrine with a significantly smaller change of both developed tension and frequency of beatings (IDT: Pt0.01;FC: PtO.O1).
620
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011
rat atrium
cytoplasm was represented by atrial granules and in the atria incubated with EVI(+) sera, the granules composed 4.2 % of the cytoplasm. Discussion In the present report, employing an hyperacute experimental design, the ability of EVI antibody to interact with the plasma membrane of living myocardial cells could be demonstrated. In addition, morphological alterations appeared in the sarcolemma of the cells in which the antibody is fixed, suggesting that these alterations are related to an action of the EVI antibody. Although bound p1 C-A was also demonstrated, classical holes as induced by the C system in other membranes (Humphrey and Dourmashkin, 1965; Anziano et al, 1972) were not observed.
The present study demonstrates that EVI positive sera can induce functional modifications in isolated atrial preparations. After brief exposures of 60 min, inotropic and chronotropic behaviour remained similar to those of EVI negative sera and controls, suggesting that the changes observed thereafter are not related to the presence of higher concentrations of pharmacologically active hormones or substances. Instead, the slow onset of the observed changes suggest a delay due to the progressive fixation of the antibodies to the membranes. In the case of cardiac anaphylaxis with isolated preparations (Feigen and Prager, 1969), histamine release and its effects on frequency and tension were observed within a few minutes. Although EVI antibodies in chronic chagasic cases are mainly IgG, so that anaphylaxis might be expected
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F I G . 8 High power view of two atrial muscle cells sarcolemma. Immediately beneath the plasma membrarte flattened cisternae can be seen (arrows). ( x 44 000.)
621 Sferin-Borda, Cossio, M . Gimeno, A . Citneno, Diez, Laguens, Meckert, and Arana
. 9 Electronmicrograph of two atrial cardiocytes. The arrow points to an area where adjacent plasmalemmae show a highly convolirted appeararice. FIG
through the activation of the complement system (Dias Da Silva and Lepow, 1967; Jensen, 1967; Miiller-Eberhard, 1968), the effects observed in the present study are not likely to be due to significant amounts of histamine release because of the slow onset of the changes. In addition, cyproheptadine did not alter the effect of EVI(+) chagasic sera. In atria beating in the media containing EVI( +) sera, a significantly increased frequency of beating was observed at 120 and 180min, whereas in those beating in media containing EVI(-) sera or in the controls the frequency diminished. This increase in frequency could be prevented by a p-adrenergic blocking agent, but not by an a-adrenergic blocking agent, or by an anti-histaminic compound. Two explanations are possible: (1) the interaction of the antibody with the specific antigen induces a slow liberation of endogeneous norepinephrine ; (2) the fixation of the EVI antibody induces a partial ‘agonist like’ effect through the Padrenergic receptor which appears to be located in the plasma membrane (Lefkowitz, 1973). The finding that tension was not substantially
modified by EVI( i - )sera remains unexplained. Both tension and frequency response changed in parallel when exogenous norepinephrine was added to the medium containing EVI( +) sera, suggesting that the antibody also had at least some effect on tension. However, at present, it is not easy to ascertain whether the smaller percentage of changes of FC following norepinephrine, observed in atria subjected to the presence of EVI(+) sera, is due to a diminished influenced of the neurotransmitter over pacemaker cells, directly associated to the antibody, or to the result that control frequency values before norepinephrine were not already higher in this group. However, this is not the case regarding the smaller inotropic reactivity towards this agent because control magnitudes of IDT before its addition were similar. Therefore, it would appear to support the existence of an effect of the antibody upon atrial peak tension, at least with respect to the effect of exogenous norepinephrine. The present results may be explained if the EVI antibody acted as a partial p-adrenergic agonist that could both increase frequency beating and also to some extent block
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( x 16 000.)
622 In-vitro effect of chagasic sera on rat atrium
The authors wish to express their thanks to Dr Arnold M. Katz and Dr Deborah Doniach for their advice and for revising and improving the English manuscript. The valuable technical assistance of M. B. Casanova is gratefully acknowledged.
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the response of the p-receptors to exogenous norepinephrine. A similar pattern of activation of biological mechanisms has been described in the case of LATS which is acting through the reaction of antibodies with membrane TSH receptors (Lancet, 1974; Smith and Hall, 1974). In this case, the evidence was obtained by a competition radioimmunoassay (Hall et a/, 1975). The EVI antibody, also reacting with plasma membranes of striated muscle (Cossio er a/, 1974a), does not have species-specificity (Cossio et a/, 1974b) and has biological activity. It cannot be established whether the EVI antibody acts directly through a specific reaction with antigenic determinants related to the p-adrenergic receptor itself, or, whether these effects arise from indirect mechanisms due to an interaction with the plasma membrane of the myocardial cells. The present report demonstrates that EVI antibody can interact with living myocardial cells to induce both morphological and functional changes. These effects appear to be related predominantly to the plasma membrane of myocardial cells. This finding is in accordance with the physiological properties of this structure, and supports the hypothetical participation of EVI antibody in some of the pathogenetic mechanisms of Chagas’ heart disease (Cossio et a / , 1974a; 1974b). Many forms of arrythmias and puzzling responses to antiarrythmic drugs and therapeutic electrical procedures are commonly found in Chagas’ heart disease; sudden cardiac death is also prominent. However, further data will be needed before the functional, pharmacological, and morphological modifications observed with the EVI antibody in our hyperacute in-vitro model can be related to the slowly developing rhythm and conduction disturbances observed in the chronic heart involvement of American trypanosomiasis.
