Endomyocardial biopsy in Chagas ' heart disease : pathogenetic contributions

Endomyocardial biopsy procedure has been performed in many centers around the world, allowing a better treatment and followup of the patients with myocardial disease. In Chagas' disease, it has been performed in Sao Paulo Heart Institute since 1978 and has brought important contributions to the understanding of the disease and consequently of the patient's clinical stage. In the present work we summarize the principal findings regarding the pathogenesis of Chagas' disease obtained mainly from the studies using endo'myocardial biopsy specimens. Nowadays we do not have doubts that the inflammatory infiltrate aggressing myocardial fibers has fundamental role in the progression of the myocardial damage in Chagas' disease what culminates in chronic heart failure. The parasite seems to have active participation in the maintenance of such myocardial inflammation.


INTRODUCTION
T he low levels of morbidity and mortality of endomyocardial biopsy technique have made very common its use in several conditions.This technique was described by Konno e Sakakibara in 1963 (10) and the main indication has been the detection earliest as possible acute rejection episodes in the follow-up of heart transplanted patients.Frequently, more than 20 consecutives endomyocardial biopsies are performed in a same patient (17).It has also being used for the diagnosis and control of the treatment of myocarditis.Employment of the endomyocardial biopsy has provided many important contributions in the understanding of the pathogenesis of chronic Chagas' disease and it may be, in the future, an important auxiliary procedure in the monitorization of the treatment of chronic chagasic patients.Palacios -Prti et al. (16), quantified the myocardial ultraestructural alterations and observed an increase of such lesions from grade I-A untill grade II.There was a mild decrease of the lesions in the grade III.
Pereira-Barreto et aI., in 1986 (18), studied endomyocardial biopsy specimens form chronic chagasic patients in different clinical stages (indeterminate, cardiac form without heart failure and cardiac form with heart failure) and demonstrated that hypertrophy and fibrosis were more frequent and more severe ,in the group of patients with heart failure.The inflammatory infiltrate was present in nearly 65% of the patients with electrocardiographic alterations (with or without heart failure) and in 37% of the patients in the indeterminate form.Higuchi et aI.( 6), studied endomyocardial biopsy specimens from the same groups of patients looking for active myocarditis.
In this work the authors used as criterium of active myocarditis the presence of lymphocytes surrounding myocardial fibers which were interpreted as foci of myocardial aggression.They concluded that myocarditis is more frequent and more severe in the group with chronic heart failure (92%), secondly in the group without heart failure (62%) and rarely in the indeterminate form (15%).
All of these findings reinforced the idea that the inflammation, mainly attacking the myocardial fibers, has a fundamental role in the evolution of the chronic chagasic cardiopathy to heart failure.The clinical evolution of the disease would represent a continuous progression of myocardial fibers destruction, substitution by fibrosis, compensatory hipertrophy, cardiac dilatation and heart failure.
The active participation of the inflammatory process in the pathogenesis of chronic cardiac Chagas' disease was contested in the past (9).However, nowadays, most of the investigators believe that immunological reactions should be involved in this process.The discrepancy between presence of parasites and intensity of inflammatory infiltrate and the difficulty to find T. cruzi parasites originated many autoimmune or hipersensitivity theories.
The finding of circulating autoantibodies in chronic chagasic patients suggested participation of humoral autoimmune phenomenon (3).Corroborating this idea Molina et aI.( 14), in 1984, detected IgG, IgA, IgM and C3 in the interstitium, capilar, endocardium and surface of cardiac fibers using endomyocardial biopsy in chronic chagasic patients, and direct immunofluorescent and immunohistochemical studies.However, Higuchi et aI.(8) did not any find immunoglobulins or complement in the myocardium of endomyocardial biopsy specimens from chronic chagasic patients presenting heart failure, also using immunofluorescence technique contradicting the idea of direct participation of autoantibodies in the pathogenesis of chronic chagasic myocarditis.
The involvement of cellular autoimunity was investigated by Higuchi et aI.(7), through the detection of which subsets of T cells were participating in chronic chagasic myocarditis and comparing with the myocardial rejection process in non-chagasic heart transplanted patients.The authors considered that the rejection could be seen as an autoimmune proces,s, since it is a reaction of the lymphocytes against myocardial fibers which are considered as non-self.The myocardial rejection process was characterized by similar numbers of CD8+ and CD4+ T cells.In contrast, the chronic chagasic myocarditis was characterized as a T cell myocarditis, mainly by CD8+ and scarce and weakly stained CD4+ cells.The authors suggested participation of the parasite inducing weak expression of CD4 T cells experimental acute infection since it is known experimentally that T. cruzi is able to decrease the expression of surface CD molecules, causing a immunodepression in the host (20).
In spite of some experimental works have suggested the possibility of development of chronic cardiopathy only injecting fragments of the parasite and consequently the disease to be associated to an auto-immune process (21), the real significance of the parasite in the pathogenesis of chronic lesions of Chagas' disease is demanding more studies.Recently we studied this matter in autopsy material (5), using policlonal anti-:r.cruzi antigen.We demonstrated a direct association betweeen presence of:r.cruzi antigens and presence of moderate or severe myocardial inflammation.The:r.cruzi were seen as rounded structures similar to degenerated amastigotes in the interstitial matrix (Fig. 2), amorphous material into the citoplasm of the macrophages (Fig. 1), or as amastigotes into myocardial fibers or endothelial cells.However, the number of parasites was generally scarce that it did not justify the intensity of myocardial inflammation, suggesting that, in parallel to the action of the parasites, other processes as autoimmunity or hipersensitivity should be participating.The presence of:r.cruzi structures outside of the cells, in our oppinion, is a very important stimulus for development of active myocarditis episodes, including aggression of non-infected myocardial fibers (auto-aggression).This finding corroborate previous thesis of hipertensitivity of many authors decades before.Magarino Torres, in 1929 (13), proposed the occurrence of hipersensitivity phenomenons induced by antigenic products released by degenerated parasites, to explain the cardiac inflammatory lesions.Muniz e Azevedo in 1947 (15), produced inflammatory lesions in hearts of monkeys using successive injections of :r.(1) produced chronic myocarditis in guinea-pigs injecting homogenates of heart associated to :r. cruzi antigen and Freund adjuvant, concluding that hipersensitivity had a fundamental role in the pathogenesis of this myocarditis.Franco, in 1972 (4), analysed acute and chronic infection in guinea-pigs and verified presence of :r.cruzi antigens in macrophages during al~o the chronic stage of the disease, suggesting that the persistence of these antigens in interstitial macrophages would induce the hipersensitivity reactions.Another data favouring the concept of chronic influence of the parasite comes from the observation of chronic chagasic patients submitted to heart transplantation.These patients frequently present episodes of reacti vation of the Chagas' di sease after immunosuppression (19) indicating that the parasite remains in constant immunological control in chronic phase of the disease.Than, if myocarditis occurs in the endomyocardial biopsies of these patients, it is necessary to search T. cruzi antigens at the myocardial sections in order to differentiate myocardial rejection process from chronic chagasic myocarditis, because both processes have the same histological feature.In case of Chagas' disease reactivation, treatment with specific antiparasitic drug determinates a fast disappearance of the inflammatory infiltrate.After all these findings we are convinced that parasite has significant influence in determining the active chronic myocarditis and that the endomyocardial biopsy may have important role in the follow up ofthese patients.In our oppinion, chronic chagasic patient with moderate .. ' .... .>::. ~/:.": : •.. , .•.: ......•.• ' .

Figure 2 .
Figure 2. Histopathological aspect from another human case of chronic chagasic myocarditis showing three degenerated T. cruzi amastigotes (through immunoperoxidasis technique) near to the sarcolemma of a myocardial fiber surrounded by moderate mononuclear inflammatory infiltrate.(IPX 400x)